Abstract
Genetic alterations of the mixed-lineage leukemia (MLL) gene are commonly identified in acute leukemias. In acute myeloid leukemia (AML), a partial tandem duplication (PTD) of MLL occurs in about 5-10% of AML patients and is associated with adverse prognosis. The mutation leads to an in-frame duplication of exons 5 to 11 resulting in the production of an aberrant MLL protein. Unlike chromosomal rearrangements of MLL, this mutation does not affect the functional histone 3 lysine 4 (H3K4) methyltransferase domain. However, AMLs carrying a MLL-PTD and MLL-rearranged leukemias share some common characteristics, such as overexpression of HOXA-cluster genes and dysregulated epigenetic functions. Recently, leukemias with various MLL-translocations have been shown to be dependent on the histone 3 lysine 79 (H3K79) methyltransferase, DOT1L, and are sensitive to EPZ004777, a recently described selective small-molecule DOT1L inhibitor. EPZ-5676, a DOT1L-inhibitor with improved potency and drug-like properties, has recently been identified and is currently under clinical investigation.
To evaluate the therapeutic potential of DOT1L-inhibition in MLL-PTD positive leukemia cells, we assessed the effect of EPZ004777 on the MLL-PTD containing leukemia cell lines MUTZ-11 and EOL-1. In vitro treatment with EPZ004777 over a 14-day period resulted in dramatic reduction of cell proliferation compared to DMSO vehicle control in both cell lines beginning 7 days after the start of treatment. Similar results were obtained for MOLM-13, a leukemia cell line harboring a MLL-translocation, but not for HL-60, a non-MLL-rearranged leukemia cell line. To further investigate whether these findings reflect a selective response to EPZ004777 or non-specific drug toxicity, we first explored the genome-wide H3K79 dimethylation (H3K79me2) profile using chromatin immunoprecipitation (ChIP) followed by next generation sequencing in untreated MUTZ-11 cells. Across the HOXA-cluster locus, we detected a similar H3K79me2 distribution pattern as previously reported in MLL-rearranged leukemias. Further analysis of H3K79me2 in MUTZ-11 and EOL-1 cells after treatment with the inhibitor showed profound suppression of those marks as assessed by western blot and ChIP-PCR. Subsequent global gene expression analysis revealed concurrent downregulation of HOXA-cluster and other MLL-target genes after 7 days of DOT1L inhibition. To investigate the effect of EPZ004777 on the MLL-PTD positive cells in more detail, we analyzed cell differentiation and apoptosis upon a 10-day exposure to the compound. As previously described for EPZ004777-sensitive MLL-rearranged leukemias, drug treatment resulted in increased expression of CD11b and morphological changes consistent with myeloid cell differentiation. We further observed apoptotic cell death after EPZ004777 treatment as measured by an increase in the percentage of Annexin V positive cells and cleaved Caspase 3 protein compared to vehicle controls. In order to determine the effect of DOT1L inhibition in vivo, we tested the recently identified DOT1L-inhibitor, EPZ-5676, for its ability to inhibit leukemia growth in a subcutaneous EOL-1 xenograft model in immunocompromised rats. Similar to what we observed in vitro, continuous intravenous administration over 21 days led to substantial dose-dependent inhibition of tumor growth, abrogation of H3K79me2, and concurrent downregulation of selected MLL-target genes.
Thus, we demonstrate unexpected sensitivity of MLL-PTD containing leukemia cell lines to the DOT1L inhibitors EPZ004777 in vitro and EPZ-5676 in vivo. These data suggest that patients with myeloid malignancies carrying this particular mutation might benefit from treatment with therapeutic approaches that target DOT1L.
Disclosures:
Daigle: Epizyme, Inc: Employment, Equity Ownership. Olhava:Epizyme Inc.: Employment. Pollock:Epizyme Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other.
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