Interrogating DOT1L Recruitment by MLL-Fusion Proteins MLL-AF9 and MLL-ENL Towards the Development of Novel Targeted Therapy

Abstract

The Mixed Lineage Leukemia (MLL) is a Trithorax (Trx) transcriptional regulator often implicated in leukemic oncogenesis. MLL1 rearrangement leukemia is a very aggressive form of leukemia that results from a translocation of chromosome 11q23. The translocation gives rise to a chimeric protein that consists of the N-terminus MLL and 1 of more than 80 fusion partners. The most common fusion partners are ENL, AF9, and AF4. ENL and AF9 are a part of the YEATS family of proteins, containing an N-terminal histone acetylation reading YEATS domain and a C-terminal ANC1 homology domain (AHD). AF9 and ENL’s AHD recruit either the Super Elongation Complex (SEC) or the histone 3 lysine 79 (H3K79) histone methyltransferase (HMT) disrupter of telomeric silencing 1-like (DOT1L) to activate gene transcription. Recruitment of DOT1L proved to be essential for the transforming activity of multiple MLL fusion proteins. Our lab mapped the 10 amino acid (865 – 874) binding site on DOT1L, which binds to the AHD domain of AF9/ENL. Applying alanine mutagenesis studies, a point mutation, I867A, was identified sufficient to disrupt the AF9-DOT1L interaction in vitro and demonstrated that DOT1L lacking the 10 amino acids (Δ10) was unable to support transformation by MLL-AF9. In this study, we used a genetic approach to explore the role of DOT1L recruitment in leukemogenesis and normal hematopoiesis to further validate the disruption of the AF9-DOT1L and ENL-DOT1L protein-protein interactions (PPI) as a potential therapeutic approach. We demonstrate that disrupting the AF9-DOT1L PPI inhibits leukemic cell growth, downregulates HOXA9 and MEIS1 gene expression, leading to cell differentiation and inducing apoptosis. These observed effects were similar to enzymatic inhibition. Nevertheless, PPI deficient adult hematopoietic cells completely reconstituted the bone marrow of mice; whereas, cells lacking DOT1L or its catalytic activity were not. We successfully designed a DOT1L peptidomimetic with a KD of 10 nM against AF9 and 25 nM to ENL that is cellularly active and selective for MLL-AF9 transduced murine cells over a non-DOT1L dependent, E2A-HLF, cells. These results emphasize the critical role of the AF9-DOT1L PPI in leukemic cell growth, but not for adult hematopoiesis making it an attractive therapeutic approach for MLL-rearrangement leukemia. Due to the homology between the AHD domain of AF9 and ENL, we utilized the same genetic approach as with MLL-AF9 to determine if blocking DOT1L recruitment in MLL-ENL cells would yield the same effect on leukemogenesis. We showed that blocking DOT1L recruitment is not sufficient to fully inhibit leukemic cell growth. We postulate that this is due the retention of the YEATS domain in the MLL-ENL fusion that is lost in MLL-AF9. We characterized two interactions that could be contributing to leukemogenesis, Paf1-YEATS, and YEATS-H3. We demonstrate that Paf1 directly interacts with ENL YEATS with a binding affinity of 15 nM and confirm the YEATS-H3K27ac interaction with a binding affinity of 80 M. We developed a fragment-based screening method using DSF to identify compounds that bind to the YEATS domain. The development of these tools will allow us to probe both the YEATS-Paf1 and YEATS-H3 interactions to determine which interactions are critical for MLL-ENL driven leukemia. Overall, these studies show the characterization of several PPIs involved in MLL-AF9/ENL leukemia and the development of tools to further elucidate their roles in leukemic and non-leukemic contexts towards novel therapy.