MLL Fusion Proteins Directly Regulate a Small Set of Wild Type MLL Target Genes.

Abstract 1279 Poster Board I-301 The MLL (mixed-lineage leukemia) gene at chromosome band 11q23 is rearranged frequently in AML and ALL, and associated with poor prognosis. The consequence of these translocations is the formation of a chimeric oncogenic transcription factor that specifies a unique expression signature distinct from other subtypes of acute leukemia. However, it is poorly understood, which changes in gene expression in leukemic cells are under the direct control of MLL fusion proteins (fusion), nor is it clear what is the potential overlap between MLL wild type (WT) and fusion target genes. In the present study, we used genome-wide location analysis to determine the genomic loci that are specifically bound by MLL fusion proteins. Combining the binding analysis with expression profiling, we further defined the subset of MLL fusion-bound genes whose expression is regulated by the presence of MLL fusion proteins. Using ChIP-chip (Chromatin Immunoprecipitation coupled with micro-array), we determined the MLL-bound regions in 5 myeloid leukemic cell lines using a custom array containing the entire genomic region of 200 genes previously found to have altered expression in MLL-rearranged leukemias. Examination of these 200 genomic loci revealed a largely overlapping set of genes bound by MLL (wild type and/or fusion proteins) in WT/WT (U937: 110 genes, HL60: 79 genes) and WT/Fusion cells (MV4;11: 62 genes, THP-1: 89 genes). Surprisingly, the MLL-bound genes in fusion/fusion (ML-2) cells (25 genes) are a small subset of that found in each of the other 4 cell lines, despite comparable levels of detected MLL binding signal across all lines examined. These data suggest that the MLL fusion protein is likely only localized to a limited portion of genomic loci occupied by the MLL wild type protein. To test this hypothesis in a more systematic way, we examined an inducible MLL-ENL–ER transformed cell line (Slany et al, MCB 2004), which grow as myeloblastic cells in the presence of MLL-ENL, and differentiate into neutrophils upon inactivation of the fusion protein. As MLL-ENL promotes histone H3 lysine 79 (H3K79) methylation, we determined both MLL binding and H3K79 methylation using a genome-wide location analysis. We anticipated that MLL-fusion bound genomic regions would exhibit a significant drop in either MLL and/or K79 signal upon inactivation of MLL-ENL. Unexpectedly, among thousands of genes that are bound by MLL, only 10% of them (222 genes) showed a pattern of binding increase between MLL-ENL induced and un-induced conditions. To explore the impact of MLL fusion protein on gene activation, we performed whole genome expression profiling in the presence or absence of MLL-ENL. Increased levels of either MLL binding or H3K79 methylation are significantly associated with differential gene expression. Among 222 MLL fusion target genes, 12 of them are differentially up-regulated in the presence of MLL-ENL, indicating that a large fraction of MLL fusion bound genes do not exhibit significant changes in mRNA expression. The identified 12 genes include key regulators in cellular differentiation and cell cycle regulation, as well as Meis1, Hoxa9 which are known to be essential for the development of MLL leukemia. To explore the apparent discrepancy between the massive expression changes in MLL rearranged leukemia and the small number of direct fusion target genes we identified, we tested the hypothesis that a significant portion of the MLL fusion protein expression signature was derived from its direct fusion target genes Meis1 and Hoxa9. Using publicly available data, we compared the MLL leukemia associated expression profile with the set of genes that were down-regulated upon knock-down of Meis1 and Hoxa9. We found significant enrichment of Hoxa9/Meis1 downstream targets in the expression profile defined by MLL fusion proteins. Altogether, our data suggest that MLL fusion proteins are likely to contribute to the development of acute leukemia through direct activation of a very small set of genes. The results have important implications in understanding the mechanisms of target gene specificity involving oncogenic transcription factors. Disclosures No relevant conflicts of interest to declare.