Demonstration of a Role for Dot1l In MLL-Rearranged Leukemia Using a Conditional Loss of Function Model
Authors: Bernt Kathrin M , Zhu Nan , Faber Joerg , Punt Natalie , Pollock Roy M , Richon Victoria M , Kung Andrew L. , Armstrong Scott 
Published: October 2019

Abstract
Abstract 62
Leukemias associated with translocations of the Mixed Lineage Leukemia (MLL) gene account for a significant percentage of both AML and ALL, and often carry a poor prognosis. The exact molecular mechanisms by which MLL-fusion proteins transform cells are incompletely understood. One proposed model involves the aberrant activation of transcriptional programs through epigenetic changes that ultimately lead to leukemogenesis. The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been shown to be recruited by the most common MLL fusion proteins, and MLL fusion protein target loci are associated with H3K79 methylation (H3K79me2/3) in mouse models and MLL-rearranged human leukemia samples. However, it is currently unclear whether H3K79 methylation is indeed a necessary step in leukemogenesis.
We sought to investigate in detail the importance of Dot1l in MLL-fusion mediated leukemia, using an shRNA approach and a newly developed conditional loss of function mouse model of Dot1l (Dot1lflox/flox). shRNA mediated suppression of Dot1l in a panel of MLL-rearranged human leukemia cell lines led to a decrease in growth rate and viability, induction of apopotosis and cell cycle arrest. Bioluminescent in vivo tracking of MLL-rearranged human leukemia cell lines in xenotransplant recipients revealed that the onset of leukemia was significantly delayed after Dot1l suppression. To complement and confirm the sh-RNA results, we developed a conditional loss of function mouse model for Dot1l. In this model, deletion of the active site of Dot1l severely impaired or abrogated serial replating of Mll-Af9 transduced lineage negative (lin-) cells, and fully developed Mll-Af9 leukemia cells. Immunoblot of total cellular H3K79 and chromatin immunoprecipitation of known MLL target loci showed loss of H3K79 methylation. In addition, we observed variable induction of additional silencing mechanism such as Polycomb Repressor Complex 2 (PRC2) mediated H3K27 methylation on selected loci such as HoxA10. These epigenetic changes correlated with a reduction in expression of known MLL-fusion downstream targets. When Dot1l was inactivated in Mll-Af9 leukemia cells, in vitro colony and cell morphology demonstrated loss of blast-like phenotype and induction of differentiation. Furthermore, mice transplanted with Mll-Af9 leukemia cells from primary recipients failed to induce leukemia in secondary recipients after cre-mediated deletion of Dot1l.
The role of Dot1l in normal hematopoiesis is not defined, and it is possible that deletion of Dot1l induces catastrophic collapse of the entire hematopoietic system, including leukemia cell compartments. To exclude this possibility, we are analyzing the phenotype of hematopoiesis specific deletion of Dot1l in normal mice through the use of the Vav-cre system. Initial results show that Dot1lflox/flox-Vav-Cre mice are born at mendelian ratios and display neutrophil and lymphocyte counts at the lower limit of normal despite near complete absence of H3K79 methylation in peripheral blood leukocytes. A more detailed analysis of the hematopoietic phenotype of loss of Dot1l, including hematopoietic stem cell compartments, is currently ongoing in our laboratory.
These data demonstrate that Dot1l is indeed a central player in MLL-fusion mediated leukemogenesis and required for leukemia cell survival. The low-normal Dot1l-/- neutrophil and lymphocyte counts observed in the Dot1lflox/flox-Vav-Cre mice suggest that Dot1l is more critical for leukemia cells than normal hematopoietic cells. This predicts a therapeutic window for pharmacologic inhibitors of Dot1l, and highlights their potential as targeted therapy for MLL-rearranged leukemias.

Disclosures:
Pollock: Epizyme, Inc: Employment. Richon:Epizyme, Inc: Employment. Armstrong:Epizyme, Inc: Consultancy.