The results presented here demonstrate that MLL-AF9 myeloid leukemia is driven by an aberrant epigenetic program involving H3K79 methylation. A potential role for Dot1l in MLL
-rearranged leukemia is supported by multiple publications in recent years. However, the contribution of Dot1l and H3K79 methylation with respect to other hypotheses of MLL-fusion driven leukemogenesis, and the importance of H3K79me2 in the maintenance of gene expression remained unclear. H3K79 methylation has been reported to be near ubiquitously coupled to transcription (Steger et al., 2008
), a fact that has raised serious concerns about the specificity of the dependence of MLL
-rearranged leukemia cells on Dot1l. It was also unclear if a single and relatively abundant histone modification could exert locus specific effects on a highly selective gene set that controls the MLL-fusion associated leukemogenic transcription program.
We demonstrate an absolute dependence of MLL-AF9 leukemia cells on Dot1l in vitro and in vivo, and propose a molecular rationale for this finding by identifying a distinct epigenetic lesion specifically involving H3K79me2. We identified the direct targets of the MLL-AF9 fusion protein in a murine MLL-AF9 leukemia model, allowing us to assess chromatin states specifically at MLL-AF9 targets. We discovered highly abnormal H3K79me2 patterns at MLL-AF9 target loci, which were unique both with respect to other transcriptionally active loci within the same cell, and with respect to the same loci when expressed at the same level but under physiologic regulation in normal hematopoietic progenitors. These abnormalities could explain a specific and non-physiological dependence of the MLL-fusion driven leukemogenic transcription program on H3K79 methylation. Consistent with this hypothesis, only a small subgroup of genes, which was highly enriched for MLL-fusion targets and other genes with known functions in MLL-rearranged leukemias, showed any change in expression after loss of Dot1l and H3K79 methylation. This group of genes showed significant overlap with gene expression signatures that define murine MLL-AF9 leukemia stem cells and human MLL-rearranged clinical leukemia samples. On the other hand, transcription of the majority of genes associated with H3K79me2 did not depend on the presence of this chromatin mark. While our studies do not address the role of H3K79 methylation with respect to initiation or elongation, they do suggest that H3K79me2 is not uniformly involved in maintaining transcription. Rather, the exquisite and specific sensitivity of MLL-AF9 leukemia cells to loss of H3K79 methylation may be a consequence of the uniquely abnormal H3K79 methylation patterns at key loci. It is of tremendous interest to determine whether the dependence of MLL-fusion associated expression programs constitutes an over-reliance on an inappropriately targeted, but otherwise normal Dot1l, or whether Dot1l is aberrantly regulated in these leukemias.
Our data support a role for Dot1l in normal hematopoiesis. Conditional Dot1l knockout mice expressing Cre from a hematopoiesis specific promoter exhibit a moderate reduction in white blood cells, and a moderate to severe reduction in red blood cells as well as earlier progenitor compartments. This phenotype is similar to the hematopoietic phenotype recently reported in a conditional Dot1l CreER model (Jo et al., 2011
). Analysis of embryonic blood development in Dot1l mutant mice revealed a severe defect in early erythroid, but not myeloid development (Feng et al., 2010
). Earlier studies support a role for Dot1l in the transcriptional regulation of the alpha and beta globin loci (Fu et al., 2005
; Sawado et al., 2008
). Taken together, these studies provide evidence for a critical role of Dot1l in early hematopoietic and particularly erythroid development, therefore anemia or pancytopenia may be potential side effects of pharmacologic inhibition of Dot1l. However, complete deletion of c-kit results in severe anemia, bone marrow failure and embryonic lethality, while clinically effective pharmacologic inhibition of c-kit with imatinib is very well tolerated with only modest and manageable hematopoietic side effects (Broxmeyer et al., 1991
). The fact that the isolation of near uniformly Dot1l−/−
peripheral blood leukocytes from 3–6 week old mice is possible demonstrates that Dot1l is not absolutely required for all hematopoietic cells, and suggests an exploitable therapeutic window.
Dot1l is the only known methyltransferase for H3K79 and responsible for mono, -di and trimethylation of this residue. Mono- di and tri methylation may fulfill divergent biological roles. Genome occupancy of H3K79me2 and H3K79me3 modified histones in yeast is mutually exclusive, and associated with distinct biological processes (Schulze et al., 2009
). Our study assessed only the genome wide methylation patterns for H3K79me2 due to lack of specific antibodies that recognize H3K79me3. Our study does not address whether loss of mono, di- or trimethylation is the most detrimental to MLL
-rearranged leukemia cells. This question is not trivial, since the addition of methyl groups by Dot1l appears to be sequential and the conversion of H3K79me2 to H3K79me3 requires prior ubiquitination of histone H2B, hinting at a much more complex epigenetic network potentially involving additional histone modifications.
In summary, our findings demonstrate a strong rationale and imply a therapeutic window for pharmacologic inhibition of Dot1l as a strategy to target MLL-leukemias. Further studies will determine the exact molecular mechanisms of how abnormal H3K79 methylation patterns are established, and how mis-targeting of a single epigenetic modification may act as a master regulator of transcriptional programs that lead to leukemic transformation.