Elucidating the Importance of DOT1L Recruitment in MLL-AF9 Leukemia and Hematopoiesis.

MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute Dot1l inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9-DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.

PAF1 complex interactions with SETDB1 mediate promoter H3K9 methylation and transcriptional repression of Hoxa9 and Meis1 in acute myeloid leukemia.

The Polymerase Associated Factor 1 complex (PAF1c) is an epigenetic co-modifying complex that directly contacts RNA polymerase II (RNAPII) and several epigenetic regulating proteins. Mutations, overexpression and loss of expression of subunits of the PAF1c are observed in various forms of cancer suggesting proper regulation is needed for cellular development. However, the biochemical interactions with the PAF1c that allow dynamic gene regulation are unclear. We and others have shown that the PAF1c makes a direct interaction with MLL fusion proteins, which are potent oncogenic drivers of acute myeloid leukemia (AML). This interaction is critical for the maintenance of MLL translocation driven AML by targeting MLL fusion proteins to the target genes Meis1 and Hoxa9. Here, we use a proteomics approach to identify protein-protein interactions with the PAF1c subunit CDC73 that regulate the function of the PAF1c. We identified a novel interaction with a histone H3 lysine 9 (H3K9) methyltransferase protein, SETDB1. This interaction is stabilized with a mutant CDC73 that is incapable of supporting AML cell growth. Importantly, transcription of Meis1 and Hoxa9 is reduced and promoter H3K9 trimethylation (H3K9me3) increased by overexpression of SETDB1 or stabilization of the PAF1c-SETDB1 interaction in AML cells. These findings were corroborated in human AML patients where increased SETDB1 expression was associated with reduced HOXA9 and MEIS1. To our knowledge, this is the first proteomics approach to search for CDC73 protein-protein interactions in AML, and demonstrates that the PAF1c may play a role in H3K9me3-mediated transcriptional repression in AML.