MLL-fusion proteins induce changes in histone modifications that result in the abnormal and sustained expression of downstream oncogenic target genes. A number of recent studies have identified aberrant histone 3 lysine 79 (H3K79) methylation by the chromatin modifying enzyme DOT1L as an important epigenetic modification that sustains MLL-target gene expression. Aberrant H3K79 methylation has been shown to be necessary for oncogenic transformation mediated by a number of MLL-fusions. These recent findings have generated tremendous interest in H3K79 methylation as a therapeutic target in the MLL rearranged leukemias. The plant-homeodomain (PHD) and leucine zipper-containing protein AF10 biochemically interacts with DOT1L and is believed to influence H3K79 methylation. We generated conditional knockout mice in which the Dot1l-interacting octapeptide-motif leucine zipper (OM-LZ) domain of Af10 was flanked by LoxP sites. Deletion of the Af10OM-LZ domain with the Cre recombinase is predicted to abrogate the Af10-Dot1l interaction. Deletion of the Af10OM-LZ domain greatly reduced global H3K79 dimethylation as assessed by immunoblotting as well as mass spectrometry in Af10OM-LZ deleted HoxA9/Meis1a transformed cells.
Given the importance of H3K79 methylation in MLL-rearranged leukemias, we sought to assess whether the transforming activity of the MLL-AF9 fusion gene was dependent on the Af10-Dot1l interaction. Using an MLL-AF9-IRES-GFP encoding retrovirus, we established immortalized blast-colony forming cultures from mouse lineage negative Sca-1 positive/Kit positive (LSK) bone marrow cells bearing floxed Af10OM-LZ alleles. Deletion of the Af10OM-LZ domain with Cre-recombinase dramatically reduced H3K79me2 on the MLL-target genes Hoxa5-10 and Meis1, leading to downregulation of these transcripts. We performed colony-forming cell (CFC) assays from MLL-AF9 transformed cells in the presence or absence of the Af10OM-LZ allele. In the first week, Af10OM-LZ deletion profoundly impaired the blast-colony forming potential of MLL-AF9 transformed LSKs and the only clones that could serially replate in subsequent passages had escaped Af10OM-LZ excision. Af10OM-LZ deleted colonies were very small and spread-out and showed morphological features of terminal myeloid differentiation. In contrast, HoxA9/Meis1 transformed LSK cells expanded normally in the absence of the Af10OM-LZ domain. These results demonstrate that the Af10OM-LZ, much like Dot1l, is critical for the in vitro transforming activity of the MLL-AF9 fusion gene, but does not non-specifically inhibit cellular proliferation.
We then sought to investigate the potential role of the Af10OM-LZ domain in the in vivo leukemogenic activity of MLL-AF9. We generated primary MLL-AF9 leukemias from LSKs harboring floxed Af10OM-LZ alleles. Deletion of the Af10OM-LZ domain in cells explanted from the MLL-AF9 primary leukemias led to a significant increase in the disease latency in secondary recipient mice. Moreover, limiting dilution analysis of MLL-AF9 leukemias with or without the Af10OM-LZ domain demonstrated a >100 fold decrease in the frequency of leukemia initiating cells in the absence of the Af10OM-LZ domain. Microarray analysis showed that a vast majority of MLL-AF9 target genes were significantly downregulated in Af10OM-LZ deleted as compared to Af10OM-LZ wildtype MLL-AF9 leukemias. However, the Af10OM-LZ deleted cells could still eventually cause leukemia. This is intriguing given that Af10OM-LZ deletion, similar to Dot1l deletion, leads to a significant reduction in H3K79 dimethylation as well as MLL-target gene expression. A more detailed analysis of H3K79 methylation using mass spectrometry revealed that in contrast to H3K79 dimethylation, global levels of H3K79 mono-methylation were largely unchanged in Af10OM-LZ deleted cells. This suggests the residual MLL-AF9 target gene expression seen in Af10OM-LZ deleted cells is maintained by H3K79 monomethylation.
Our results demonstrate a surprising role for Af10 in the conversion of H3K79 monomethylation to dimethylation and reveal the AF10-DOT1L interaction as an attractive therapeutic target in MLL-rearranged leukemias.
Asterisk with author names denotes non-ASH members.