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MLL-AF9 and MLL-ENL alter the dynamic association of transcriptional regulators with genes critical for leukemia.
ABSTRACT: The aim of this study was to better understand how mixed lineage leukemia (MLL) fusion proteins deregulate the expression of genes critical for leukemia.The transforming domain of one of the most common MLL fusion partners, AF9, was immunopurified after expression in myeloblastic M1 cells, and associating proteins were identified by mass spectrometric analysis. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction was used to determine how binding of associating proteins compare across Hoxa9 and Meis1 in cell lines with and without MLL fusion proteins and how binding is altered during gene down-regulation and differentiation.Consistent with earlier purifications of ENL and AF4 from 293 cells, the 90 amino acid C-terminal domain of AF9 associates with many other MLL translocation partners including Enl, Af4, Laf4, Af5q31, Ell, and Af10. This complex, termed elongation assisting proteins (EAPs), also contains the RNA polymerase II C-terminal domain kinase Cdk9/Cyclin T1/T2 (pTEFb) and the histone H3 lysine 79 methyltransferase Dot1L. Myeloid cells transformed by MLL fusions show higher levels and a broader distribution of EAP components at genes critical for leukemia. Inhibition of EAP components pTEFb and Dot1l show that both contribute significantly to activation of Hoxa9 and Meis1 expression. EAP is dynamically associated with the Hoxa9 and Meis1 loci in hematopoietic cells and rapidly dissociates during induction of differentiation. In the presence of MLL fusion proteins, its dissociation is prevented.The findings suggest that MLL fusion proteins deregulate genes critical for leukemia by excessive recruitment and impaired dissociation of EAP from target loci.
Project description:Chimeric proteins joining the histone methyltransferase MLL with various fusion partners trigger distinctive lymphoid and myeloid leukemias. Here, we immunopurified proteins associated with ENL, a protein commonly fused to MLL. Identification of these ENL-associated proteins (EAPs) by mass spectrometry revealed enzymes with a known role in transcriptional elongation (RNA polymerase II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and colocalization experiments. Purified EAP showed a histone H3 lysine 79-specific methylase activity, displayed a robust RNAPolII CTD kinase function, and counteracted the effect of the pTEFb inhibitor 5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on elongation, and inhibited transient transcriptional reporter activity. According to structure-function data, DOT1L recruitment was important for transformation by the MLL-ENL fusion derivative. These results suggest a function of ENL in histone modification and transcriptional elongation.
Project description:MLL fusion proteins are oncogenic transcription factors that are associated with aggressive lymphoid and myeloid leukemias. We constructed an inducible MLL fusion, MLL-ENL-ERtm, that rendered the transcriptional and transforming properties of MLL-ENL strictly dependent on the presence of 4-hydroxy-tamoxifen. MLL-ENL-ERtm-immortalized hematopoietic cells required 4-hydroxy-tamoxifen for continuous growth and differentiated terminally upon tamoxifen withdrawal. Microarray analysis performed on these conditionally transformed cells revealed Hoxa9 and Hoxa7 as well as the Hox coregulators Meis1 and Pbx3 among the targets upregulated by MLL-ENL-ERtm. Overexpression of the Hox repressor Bmi-1 inhibited the growth-transforming activity of MLL-ENL. Moreover, the enforced expression of Hoxa9 in combination with Meis1 was sufficient to substitute for MLL-ENL-ERtm function and to maintain a state of continuous proliferation and differentiation arrest. These results suggest that MLL fusion proteins impose a reversible block on myeloid differentiation through aberrant activation of a limited set of homeobox genes and Hox coregulators that are consistently expressed in MLL-associated leukemias.
Project description:The eleven-nineteen leukemia (ENL) protein family, composed of ENL and AF9, is a common component of 3 transcriptional modulators: AF4-ENL-P-TEFb complex (AEP), DOT1L-AF10-ENL complex (referred to as the DOT1L complex) and polycomb-repressive complex 1 (PRC1). Each complex associates with chromatin via distinct mechanisms, conferring different transcriptional properties including activation, maintenance, and repression. The mixed-lineage leukemia (MLL) gene often fuses with ENL and AF10 family genes in leukemia. However, the functional interrelationship among those 3 complexes in leukemic transformation remains largely elusive. Here, we have shown that MLL-ENL and MLL-AF10 constitutively activate transcription by aberrantly inducing both AEP-dependent transcriptional activation and DOT1L-dependent transcriptional maintenance, mostly in the absence of PRC1, to fully transform hematopoietic progenitors. These results reveal a cooperative transcriptional activation mechanism of AEP and DOT1L and suggest a molecular rationale for the simultaneous inhibition of the MLL fusion-AF4 complex and DOT1L for more effective treatment of MLL-rearranged leukemia.
Project description:Mixed-lineage leukemia (MLL) is a proto-oncogene frequently involved in chromosomal translocations associated with acute leukemia. These chromosomal translocations commonly result in MLL fusion proteins that dysregulate transcription. Recent data suggest that the MYB proto-oncogene, which is an important regulator of hematopoietic cell development, has a role in leukemogenesis driven by the MLL-ENL fusion protein, but exactly how is unclear. Here we have demonstrated that c-Myb is recruited to the MLL histone methyl transferase complex by menin, a protein important for MLL-associated leukemic transformation, and that it contributes substantially to MLL-mediated methylation of histone H3 at lysine 4 (H3K4). Silencing MYB in human leukemic cell lines and primary patient material evoked a global decrease in H3K4 methylation, an unexpected decrease in HOXA9 and MEIS1 gene expression, and decreased MLL and menin occupancy in the HOXA9 gene locus. This decreased occupancy was associated with a diminished ability of an MLL-ENL fusion protein to transform normal mouse hematopoietic cells. Previous studies have shown that MYB expression is regulated by Hoxa9 and Meis1, indicating the existence of an autoregulatory feedback loop. The finding that c-Myb has the ability to direct epigenetic marks, along with its participation in an autoregulatory feedback loop with genes known to transform hematopoietic cells, lends mechanistic and translationally relevant insight into its role in MLL-associated leukemogenesis.
Project description:Leukemia stem cells (LSC) are resistant to conventional chemotherapy and persistent LSC after chemotherapy are supposed to be a major cause of relapse. However, information on genetic or epigenetic regulation of stem cell properties is still limited and LSC-targeted drugs have scarcely been identified. Epigenetic regulators are associated with many cellular processes including maintenance of stem cells. Of note are polycomb group proteins, because they potentially control stemness, and can be pharmacologically targeted by a selective inhibitor (DZNep). Therefore, we investigated the therapeutic potential of EZH2 inhibition in mixed lineage leukemia (MLL) fusion leukemia. Intriguingly, EZH2 inhibition by DZNep or shRNA not only suppressed MLL fusion leukemia proliferation but also reduced leukemia initiating cells (LIC) frequency. Expression analysis suggested that p16 upregulation was responsible for LICs reduction. Knockdown of p16 canceled the survival advantage of mice treated with DZNep. Chromatin immunoprecipitation assays demonstrated that EZH2 was highly enriched around the transcription-start-site of p16, together with H3K27 methylation marks in MLL/ENL and Hoxa9/Meis1 transduced cells but not in E2A/HLF transduced cells. Although high expression of Hoxa9 in MLL fusion leukemia is supposed to be responsible for the recruitment of EZH2, our data also suggest that there may be some other mechanisms independent of Hoxa9 activation to suppress p16 expression, because expression levels of Hoxa9 and p16 were not inversely related between MLL/ENL and Hoxa9/Meis1 transduced cells. In summary, our findings show that EZH2 is a potential therapeutic target of MLL fusion leukemia stem cells.
Project description:Abdominal-type HoxA genes in combination with Meis1 are well-documented on-cogenes in various leukemias but it is unclear how they exert their transforming function. Here we used a system of conditional transformation by an inducible mixed lineage leukemia-eleven-nineteen leukemia (MLL-ENL) oncoprotein to overexpress Hoxa9 and Meis1 in primary hematopoietic cells. Arrays identified c-Myb and a c-Myb target (Gstm1) among the genes with the strongest response to Hoxa9/Meis1. c-Myb overexpression was verified by Northern blot and quantitative reverse transcription-polymerase chain reaction (RT-PCR). Also MLL-ENL activated c-Myb through up-regulation of Hoxa9 and Meis1. Consequently, short-term suppression of c-Myb by small inhibitory RNA (siRNA) efficiently inhibited transformation by MLL-ENL but did not impair transformation by transcription factor E2A-hepatic leukemia factor (E2A-HLF). The anti c-Myb siRNA effect was abrogated by coexpression of a c-Myb derivative with a mutated siRNA target site. The introduction of a dominant-negative c-Myb mutant had a similar but weaker effect on MLL-ENL-mediated transformation. Hematopoietic precursors from mice homozygous for a hypo-morphic c-Myb allele were more severely affected and could be transformed neither by MLL-ENL nor by E2A-HLF. Ectopic expression of c-Myb induced a differentiation block but c-Myb alone was not transforming in a replating assay similar to Hoxa9/Meis1. These results suggest that c-Myb is essential but not sufficient for Hoxa9/Meis1 mediated transformation.
Project description:The MLL fusion proteins, AF9 and ENL, activate target genes in part via recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like). Here we report biochemical, biophysical, and functional characterization of the interaction between DOT1L and MLL fusion proteins, AF9/ENL. The AF9/ENL-binding site in human DOT1L was mapped, and the interaction site was identified to a 10-amino acid region (DOT1L865-874). This region is highly conserved in DOT1L from a variety of species. Alanine scanning mutagenesis analysis shows that four conserved hydrophobic residues from the identified binding motif are essential for the interactions with AF9/ENL. Binding studies demonstrate that the entire intact C-terminal domain of AF9/ENL is required for optimal interaction with DOT1L. Functional studies show that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that DOT1L interaction with MLL-AF9 and its recruitment are required for transformation by MLL-AF9. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL fusion protein-associated leukemia.
Project description:Mixed-lineage leukemia (MLL) fusions are potent oncogenes that initiate aggressive forms of acute leukemia. As aberrant transcriptional regulators, MLL-fusion proteins alter gene expression in hematopoietic cells through interactions with the histone H3 lysine 79 (H3K79) methyltransferase DOT1L. Notably, interference with MLL-fusion cofactors like DOT1L is an emerging therapeutic strategy in this disease. Here, we identify the histone H2B E3 ubiquitin ligase ring finger protein 20 (RNF20) as an additional chromatin regulator that is necessary for MLL-fusion-mediated leukemogenesis. Suppressing the expression of Rnf20 in diverse models of MLL-rearranged leukemia leads to inhibition of cell proliferation, under tissue culture conditions as well as in vivo. Rnf20 knockdown leads to reduced expression of MLL-fusion target genes, effects resembling Dot1l inhibition. Using ChIP-seq, we found that H2B ubiquitination is enriched in the body of MLL-fusion target genes, correlating with sites of H3K79 methylation and transcription elongation. Furthermore, Rnf20 is required to maintain local levels of H3K79 methylation by Dot1l at Hoxa9 and Meis1. These findings support a model whereby cotranscriptional recruitment of Rnf20 at MLL-fusion target genes leads to amplification of Dot1l-mediated H3K79 methylation, thereby rendering leukemia cells dependent on Rnf20 to maintain their oncogenic transcriptional program.
Project description:MLL-fusion proteins, AF9 and ENL, play an essential role in the recruitment of DOT1L and the H3K79 hypermethylation of MLL target genes, which is pivotal for leukemogenesis. Blocking these interactions may represent a novel therapeutic approach for MLL-rearranged leukemia. Based on the 7 mer DOT1L peptide, a class of peptidomimetics was designed. Compound 21 with modified middle residues, achieved significantly improved binding affinities to AF9 and ENL, with KD values of 15 nM and 57 nM, respectively. Importantly, 21 recognizes and binds to the cellular AF9 protein and effectively inhibits the AF9-DOT1L interactions in cells. Modifications of the N- and C-termini of 21 resulted in 28 with 2-fold improved binding affinity to AF9 and much decreased peptidic characteristics. Our study provides a proof-of-concept for development of nonpeptidic compounds to inhibit DOT1L activity by targeting its recruitment and the interactions between DOT1L and MLL-oncofusion proteins AF9 and ENL.
Project description:Disruptor of telomeric silencing 1-like (Dot1l) is a histone 3 lysine 79 methyltransferase. Studies of constitutive Dot1l knockout mice show that Dot1l is essential for embryonic development and prenatal hematopoiesis. DOT1L also interacts with translocation partners of Mixed Lineage Leukemia (MLL) gene, which is commonly translocated in human leukemia. However, the requirement of Dot1l in postnatal hematopoiesis and leukemogenesis of MLL translocation proteins has not been conclusively shown. With a conditional Dot1l knockout mouse model, we examined the consequences of Dot1l loss in postnatal hematopoiesis and MLL translocation leukemia. Deletion of Dot1l led to pancytopenia and failure of hematopoietic homeostasis, and Dot1l-deficient cells minimally reconstituted recipient bone marrow in competitive transplantation experiments. In addition, MLL-AF9 cells required Dot1l for oncogenic transformation, whereas cells with other leukemic oncogenes, such as Hoxa9/Meis1 and E2A-HLF, did not. These findings illustrate a crucial role of Dot1l in normal hematopoiesis and leukemogenesis of specific oncogenes.