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

Abstract

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.

MEIS1 in Hematopoiesis and Cancer. How MEIS1-PBX Interaction Can Be Used in Therapy

“…9.1.1. Targeting the Enzyme DOT1L (KMT4), Which Methylates Lysine 79 of Histone H3 (H3K79) DOT1L methyltransferase activity is critical to MLL-r leukemia [121] and is recruited on DNA where it induces hyperexpression of HOXA9 and MEIS1 [122]. During the past few years, inhibitors of DOT1L have shown potent and selective activity against MLL1-r leukemia [123][124][125].…”

The Role of DOT1L in Normal and Malignant Hematopoiesis

“…One study noticed anemia and bleeding in multiple organs upon Dot1l deletion ( Nguyen et al, 2011a ). Similar effects on steady-state hematopoiesis, namely BM hypocellularity and decreases in HSPC compartments, were observed in the Dot1l f/f MxCre model upon polyinosinic:polycytidylic acid-mediated DOT1L deletion in hematopoietic cells ( Grigsby et al, 2021 ). Overall, these studies point to an essential role of DOT1L in adult normal hematopoiesis by affecting HSC self-renewal and differentiation.…”

“…Grigsby et al utilized methyltransferase mutants in which the SAM-binding domain was mutated in rescue experiments in a normal hematopoiesis study. They found that WT, but not the enzymatic dead mutant, can rescue Dot1l KO HSPCs expanded by NUP98-HOXD10HD in transplant models ( Grigsby et al, 2021 ), suggesting that DOT1L methyltransferase activity is required in this context.…”

“…In addition to DOT1L’s methyltransferase activity, studies have shown that loss of Dot1 complex (DotCom, composed of AF9, AF10, ENL and AF17) components AF10 ( Deshpande et al, 2014 ), and ENL ( Wan et al, 2017 ) have a similar effect on leukemia cells as loss of DOT1L. Similarly, the DOT1L and AF9 interaction has been shown to be important for leukemogenesis ( Shen et al, 2013 ; Kuntimaddi et al, 2015 ; Grigsby et al, 2021 ). Studies of methyltransferase mutant, AF9-binding disrupted mutant, and wild type DOT1L models in vivo showed loss of DOT1L-AF9 was sufficient to inhibit leukemia cell growth and increase their differentiation to similar levels observed with DOT1L enzyme-dead mutant ( Shen et al, 2013 ; Kuntimaddi et al, 2015 ; Grigsby et al, 2021 ).…”

Gene Transcription as a Therapeutic Target in Leukemia

“…Inactivation of DOT1L significantly suppressed the HOXA9 and MEIS1 genes associated with MLL translocation and leukemogenesis, leading to decreased proliferation, increased differentiation, and apoptosis of MLL–AF9 cells, indicating its potential for AML therapy [ 37 , 65 , 97 , 99 ]. MLL fusions with proteins interacting with DOT1L (e.g., AF9, ENL, AF17, and AF10) incorrectly target DOT1L to the promoters of the HOXA genes, which leads to methylation of H3K79 and constitutive activation of these genes [ 22 , 98 , 101 , 102 ]. This makes DOT1L methyltransferase a propitious therapeutic target in leukemia.…”