Aberrant chromatin at genes encoding stem cell regulators in human mixed-lineage leukemia

Abstract

Mixed-lineage leukemia (MLL) fusion proteins are potent inducers of leukemia, but how these proteins generate aberrant gene expression programs is poorly understood. Here we show that the MLL-AF4 fusion protein occupies developmental regulatory genes important for hematopoietic stem cell identity and self-renewal in human leukemia cells. These MLL-AF4-bound regions have grossly altered chromatin structure, with histone modifications catalyzed by trithorax group proteins and DOT1 extending across large domains. Our results define direct targets of the MLL fusion protein, reveal the global role of epigenetic misregulation in leukemia, and identify new targets for therapeutic intervention in cancer.Supplemental material is available at http://www.genesdev.org.Received September 16, 2008; revised version accepted November 4, 2008. Chromosomal translocations involving the mixed-lineage leukemia gene (MLL) are a frequent occurrence in human acute leukemias of both children and adults (Eguchi et al. 2005). In over half of all infant acute leukemias, the MLL protein fuses to one of >50 identified partner genes, resulting in a MLL fusion protein that acts as a potent oncogene (Krivtsov and Armstrong 2007). While extensive gene expression signatures have been determined for primary human leukemia samples (Armstrong et al. 2002;Yeoh et al. 2002;Ferrando et al. 2003;Ross et al. 2003;Rozovskaia et al. 2003;Haferlach et al. 2005), the direct genomic targets of MLL fusion proteins remain unknown. This information is essential to determine how MLL fusion proteins impose oncogenic transcriptional programs and to identify targets for therapeutic intervention in human disease.Distinct chromatin-modifying complexes and histone modifications are associated with distinct phases of transcription (Li et al. 2007). The trithorax group proteins, including MLL, catalyze histone H3-Lys-4 trimethyl (H3K4me3) modifications at the start sites of transcriptionally engaged genes (Ruthenburg et al. 2007). These H3K4me3-modified regions are largely constrained to the transcription start site regions of genes that are transcriptionally initiated, but not necessarily fully transcribed (Bernstein et al. 2006;Barski et al. 2007;Guenther et al. 2007). As a gene becomes fully transcribed, elongating RNA Polymerase II (Pol II) molecules proceed through gene coding regions along with associated elongation factors including DOT1, which catalyzes dimethylation of histone H3-Lys-79 (H3K79me2) (Li et al. 2007). Physical interactions between the most common MLL partner proteins and transcriptional elongation components suggest that defects in H3K4 and H3K79 methylation might be a key factor in MLL leukemogenesis In order to define the portion of gene regulatory circuitry that is controlled directly by MLL fusion proteins in human leukemia, we determined the binding patterns of an MLL fusion protein and chromatin modifications across the entire human genome. We performed this mapping in leukemic cells harboring the MLL-AF4 fusion gene, because this rearran...

Prognostic significance of FLT3 mutational status and expression levels in MLL-AF4+ and MLL-germline acute lymphoblastic leukemia

“…This increased FLT3 expression in the absence of FLT3-activating mutations suggests that the constitutive overexpression of FLT3 is a result of aberrantly increased transcription of FLT3 and it is not due to FLT3-activating mutations. This is supported by Guenther et al, 19 who showed through elegant studies that FLT3 may be a direct target of MLL-AF4. We have analyzed the most common FLT3 mutations found in acute leukemia but have not sequenced the entire FLT3 coding sequence, and therefore, although unlikely, 15 the presence of novel FLT3-activating mutations responsible for constitutive activation of FLT3 might not be completely excluded.…”

“…Despite this emerging controversy, FLT3 is consistently highly expressed in MLLrearranged ALL, and therefore it is not fully resolved whether the constitutive activation of FLT3 in MLL-rearranged ALL is due to the presence of activating FLT3 mutations, or simply due to a transcriptional increased expression of FLT3 in an attempt to provide blast cells with survival and proliferative advantage. The latter has been recently suggested by Guenther et al, 19 by showing through elegant studies that FLT3 may be a direct target of MLL-AF4. 1 Interestingly, there is little information about the prognostic significance of FLT3 expression and mutational status in cytogenetically distinct subgroups of ALLs.…”

A human ESC model for MLL-AF4 leukemic fusion gene reveals an impaired early hematopoietic-endothelial specification

“…Importantly, Guenther et al [36] recently reported a list of 42 direct targets of MLL-AF4. Despite constituting different experimental approaches (non-transformed hESCs vs fully transformed leukemia cell lines), we have compared our GEP with the MLL-AF4 direct targets proposed by Guenther et al [36] and found that 8 (ERG, TNRC18, ADAM10, HOXA10, HOXA9, MEIS1, MEF2C, ZEB2) out of these 42 (20%) MLL-AF4 direct targets were also upregulated in our experimental system.…”

DOT1L Inhibitor EPZ-5676 Displays Synergistic Antiproliferative Activity in Combination with Standard of Care Drugs and Hypomethylating Agents inMLL-Rearranged Leukemia Cells

“…In this manner, DOT1L is recruited to gene locations normally under the control of MLL (Okada et al, 2005;Mueller et al, 2007;Monroe et al, 2011). DOT1L catalyzes the specific methylation of histone H3 at lysine 79 (H3K79), and this site-specific marking of histone H3 leads to transcriptional activation (Milne et al, 2005;Okada et al, 2005;Guenther et al, 2008;Krivtsov et al, 2008;Mueller et al, 2009;Thiel et al, 2010;Monroe et al, 2011;Nguyen et al, 2011). It has therefore been speculated that DOT1L enzymatic activity represents an oncogenic driver of MLL-r leukemia, and that inhibition of the DOT1L enzyme would represent a cogent approach to therapeutic intervention for MLL-r patients.…”