Abstract 3502

The t(6;11)(q27;q23) produces a chimeric MLL-AF6 oncogene, and is a recurrent chromosomal rearrangement observed in patients with diverse hematologic malignancies such as acute myelogenous leukemia (AML), as well as both B-cell and T-cell acute lymphoblastic leukemias (ALL). The presence of an MLL-AF6 translocation predicts a particularly poor prognosis. Of particular biological interest, the MLL-AF6 translocation is the most common fusion event in which MLL fuses to a predominantly cytoplasmic protein. Very little is known about the molecular mechanisms of transformation mediated by the MLL-AF6 fusion oncogene, forestalling the development of specific therapeutic strategies for t(6;11)(q27;q23) positive leukemias. Recent studies suggest that the histone methyltransferase DOT1L could be an important therapeutic target in MLL-rearranged leukemias. We sought to assess whether MLL-AF6 mediated transformation is also dependent on aberrant H3K79 methylation using genomic, genetic and pharmacological approaches. First, we performed chromatin immuno-precipitation using H3K79me2 specific antibodies followed by next generation sequencing (ChIP-seq) on murine MLL-AF6 leukemias as well as on ML2, the human myelomonocytic leukemia cell line bearing the MLL-AF6 fusion gene. We observed that in both murine and human MLL-AF6 leukemia cells, MLL-fusion target genes display markedly high levels of H3K79 dimethylation as compared to other highly expressed genes. We then investigated whether MLL-AF6-induced transformation was dependent on aberrant H3K79 methylation through genetic or pharmacologic inhibition of the Dot1l histone methyltransferase. Lineage negative/Sca-1 positive/Kit positive (LSK) cells from mice bearing homozygous Dot1l floxed alleles were immortalized by retroviral expression of the MLL-AF6 fusion gene. Cre-recombinase mediated excision of Dot1l from MLL-AF6 transformed bone marrow cells resulted in a significant reduction in H3K79 dimethylation at the promoters of the MLL-target genes Hoxa9, Hoxa10 and Meis1, with a concomitant decrease in their expression. Dot1l excision significantly diminished the clonogenic capacity, abrogated blast colony formation in methylcellulose based medium, and enhanced differentiation of MLL-AF6 transformed cells.

We then sought to assess whether EPZ004777, a recently described specific small molecule inhibitor of DOT1L could show efficacy against murine and human MLL-AF6 transformed cells. Dot1l inhibition using EPZ004777 significantly diminished H3K79 dimethylation globally (as assessed by immunoblotting) as well as on MLL-target genes (as assessed by ChIP-qPCR) using H3K79me2 specific antibodies. Importantly, EPZ004777 treatment significantly impaired the proliferation of both murine MLL-AF6 transformed cells as well as the ML2 cell line, whereas the proliferation rates of Hoxa9-Meis1 transformed cells as well as the human MLL-germline cell line HL60 were unaffected despite a similar decrease in H3K79me2 levels. EPZ004777 treatment induced cell cycle arrest as well as increased apoptosis in MLL-AF6 positive, but not control leukemia cells, demonstrating a selective activity of the DOT1L inhibitor EPZ004777 on MLL-AF6 transformed cells.

In summary, we demonstrate that the MLL-AF6 oncoprotein requires continued activity of the histone methyltransferase DOT1L for aberrant epigenetic activation of downstream target oncogenes. More studies are needed to understand the mechanisms by which DOT1L is recruited to MLL-target genes by the MLL-AF6 fusion, since AF6 is not believed to normally associate with DOT1L. Nevertheless, the demonstration that H3K79 methylation is important for MLL-AF6 mediated transformation indicates that patients bearing the t(6;11)(q27;q23) translocation may benefit from therapeutic agents targeting aberrant H3K79 methylation.


Olhava:Epizyme: Employment. Daigle:Epizyme, Inc.: Employment. Richon:Epizyme, Inc.: Employment, Equity Ownership. Pollock:Epizyme Inc.: Employment, Equity Ownership. Armstrong:Epizyme: Consultancy.

Author notes


Asterisk with author names denotes non-ASH members.