The full transforming capacity of MLL-Af4 is interlinked with lymphoid lineage commitment.

Chromosome rearrangements involving the mixed-lineage leukemia gene (MLL) create MLL-fusion proteins, which could drive both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The lineage decision of MLL-fusion leukemia is influenced by the fusion partner and microenvironment. To investigate the interplay of fusion proteins and microenvironment in lineage choice, we transplanted human hematopoietic stem and progenitor cells (HSPCs) expressing MLL-AF9 or MLL-Af4 into immunodeficient NSGS mice, which strongly promote myeloid development. Cells expressing MLL-AF9 efficiently developed AML in NSGS mice. In contrast, MLL-Af4 cells, which were fully oncogenic under lymphoid conditions present in NSG mice, displayed compromised transformation capacity in a myeloid microenvironment. MLL-Af4 activated a self-renewal program in a lineage-dependent manner, showing the leukemogenic activity of MLL-Af4 was interlinked with lymphoid lineage commitment. The C-terminal homology domain (CHD) of Af4 was sufficient to confer this linkage. Although the MLL-CHD fusion protein failed to immortalize HSPCs in myeloid conditions in vitro, it could successfully induce ALL in NSG mice. Our data suggest that defective self-renewal ability and leukemogenesis of MLL-Af4 myeloid cells could contribute to the strong B-cell ALL association of MLL-AF4 leukemia observed in the clinic.

Instructive Role of MLL-Fusion Proteins Revealed by a Model of t(4;11) Pro-B Acute Lymphoblastic Leukemia.

The t(4;11)(q21;q23) fuses mixed-lineage leukemia (MLL) to AF4, the most common MLL-fusion partner. Here we show that MLL fused to murine Af4, highly conserved with human AF4, produces high-titer retrovirus permitting efficient transduction of human CD34+ cells, thereby generating a model of t(4;11) pro-B acute lymphoblastic leukemia (ALL) that fully recapitulates the immunophenotypic and molecular aspects of the disease. MLL-Af4 induces a B ALL distinct from MLL-AF9 through differential genomic target binding of the fusion proteins leading to specific gene expression patterns. MLL-Af4 cells can assume a myeloid state under environmental pressure but retain lymphoid-lineage potential. Such incongruity was also observed in t(4;11) patients in whom leukemia evaded CD19-directed therapy by undergoing myeloid-lineage switch. Our model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis.

MLL fusion proteins preferentially regulate a subset of wild-type MLL target genes in the leukemic genome.

MLL encodes a histone methyltransferase that is critical in maintaining gene expression during embryonic development and hematopoiesis. 11q23 translocations result in the formation of chimeric MLL fusion proteins that act as potent drivers of acute leukemia. However, it remains unclear what portion of the leukemic genome is under the direct control of MLL fusions. By comparing patient-derived leukemic cell lines, we find that MLL fusion-bound genes are a small subset of that recognized by wild-type MLL. In an inducible MLL-ENL model, MLL fusion protein binding and changes in H3K79 methylation are limited to a specific portion of the genome, whereas wild-type MLL distributes to a much larger set of gene loci. Surprisingly, among 223 MLL-ENL-bound genes, only 12 demonstrate a significant increase in mRNA expression on induction of the fusion protein. In addition to Hoxa9 and Meis1, this includes Eya1 and Six1, which comprise a heterodimeric transcription factor important in several developmental pathways. We show that Eya1 has the capacity to immortalize hematopoietic progenitor cells in vitro and collaborates with Six1 in hematopoietic transformation assays. Altogether, our data suggest that MLL fusions contribute to the development of acute leukemia through direct activation of a small set of target genes.

Aberrant overexpression and function of the miR-17-92 cluster in MLL-rearranged acute leukemia.

MicroRNA (miRNA)-17-92 cluster (miR-17-92), containing seven individual miRNAs, is frequently amplified and overexpressed in lymphomas and various solid tumors. We have found that it is also frequently amplified and the miRNAs are aberrantly overexpressed in mixed lineage leukemia (MLL)-rearranged acute leukemias. Furthermore, we show that MLL fusions exhibit a much stronger direct binding to the locus of this miRNA cluster than does wild-type MLL; these changes are associated with elevated levels of histone H3 acetylation and H3K4 trimethylation and an up-regulation of these miRNAs. We further observe that forced expression of this miRNA cluster increases proliferation and inhibits apoptosis of human cells. More importantly, we show that this miRNA cluster can significantly increase colony-forming capacity of normal mouse bone marrow progenitor cells alone and, particularly, in cooperation with MLL fusions. Finally, through combinatorial analysis of miRNA and mRNA arrays of mouse bone marrow progenitor cells transfected with this miRNA cluster and/or MLL fusion gene, we identified 363 potential miR-17-92 target genes that exhibited a significant inverse correlation of expression with the miRNAs. Remarkably, these potential target genes are significantly enriched (P < 0.01; >2-fold) in cell differentiation, hematopoiesis, cell cycle, and apoptosis. Taken together, our studies suggest that overexpression of miR-17-92 cluster in MLL-rearranged leukemias is likely attributed to both DNA copy number amplification and direct up-regulation by MLL fusions, and that the miRNAs in this cluster may play an essential role in the development of MLL-associated leukemias through inhibiting cell differentiation and apoptosis, while promoting cell proliferation, by regulating relevant target genes.

Consistent deregulation of gene expression between human and murine MLL rearrangement leukemias.

Important biological and pathologic properties are often conserved across species. Although several mouse leukemia models have been well established, the genes deregulated in both human and murine leukemia cells have not been studied systematically. We performed a serial analysis of gene expression in both human and murine MLL-ELL or MLL-ENL leukemia cells and identified 88 genes that seemed to be significantly deregulated in both types of leukemia cells, including 57 genes not reported previously as being deregulated in MLL-associated leukemias. These changes were validated by quantitative PCR. The most up-regulated genes include several HOX genes (e.g., HOX A5, HOXA9, and HOXA10) and MEIS1, which are the typical hallmark of MLL rearrangement leukemia. The most down-regulated genes include LTF, LCN2, MMP9, S100A8, S100A9, PADI4, TGFBI, and CYBB. Notably, the up-regulated genes are enriched in gene ontology terms, such as gene expression and transcription, whereas the down-regulated genes are enriched in signal transduction and apoptosis. We showed that the CpG islands of the down-regulated genes are hypermethylated. We also showed that seven individual microRNAs (miRNA) from the mir-17-92 cluster, which are overexpressed in human MLL rearrangement leukemias, are also consistently overexpressed in mouse MLL rearrangement leukemia cells. Nineteen possible targets of these miRNAs were identified, and two of them (i.e., APP and RASSF2) were confirmed further by luciferase reporter and mutagenesis assays. The identification and validation of consistent changes of gene expression in human and murine MLL rearrangement leukemias provide important insights into the genetic base for MLL-associated leukemogenesis.

Distinct microRNA expression profiles in acute myeloid leukemia with common translocations.

MicroRNAs (miRNAs) are postulated to be important regulators in cancers. Here, we report a genome-wide miRNA expression analysis in 52 acute myeloid leukemia (AML) samples with common translocations, including t(8;21)/AML1(RUNX1)-ETO(RUNX1T1), inv(16)/CBFB-MYH11, t(15;17)/PML-RARA, and MLL rearrangements. Distinct miRNA expression patterns were observed for t(15;17), MLL rearrangements, and core-binding factor (CBF) AMLs including both t(8;21) and inv(16) samples. Expression signatures of a minimum of two (i.e., miR-126/126*), three (i.e., miR-224, miR-368, and miR-382), and seven (miR-17-5p and miR-20a, plus the aforementioned five) miRNAs could accurately discriminate CBF, t(15;17), and MLL-rearrangement AMLs, respectively, from each other. We further showed that the elevated expression of miR-126/126* in CBF AMLs was associated with promoter demethylation but not with amplification or mutation of the genomic locus. Our gain- and loss-of-function experiments showed that miR-126/126* inhibited apoptosis and increased the viability of AML cells and enhanced the colony-forming ability of mouse normal bone marrow progenitor cells alone and particularly, in cooperation with AML1-ETO, likely through targeting Polo-like kinase 2 (PLK2), a tumor suppressor. Our results demonstrate that specific alterations in miRNA expression distinguish AMLs with common translocations and imply that the deregulation of specific miRNAs may play a role in the development of leukemia with these associated genetic rearrangements.

ELL and EAF1 are Cajal body components that are disrupted in MLL-ELL leukemia.

The (11;19)(q23;p13.1) translocation in acute leukemia results in the formation of a chimeric MLL-ELL fusion protein. ELL is an RNA Polymerase II (Pol II) transcriptional elongation factor that interacts with the recently identified EAF1 protein. Here, we show that ELL and EAF1 are components of Cajal bodies (CBs). Although ELL and EAF1 colocalize with p80 coilin, the signature protein of CBs, ELL and EAF1 do not exhibit a direct physical interaction with p80 coilin. Treatment of cells with actinomycin D, DRB, or alpha-amanitin, specific inhibitors of Pol II, disperses ELL and EAF1 from CBs, indicating that localization of ELL and EAF1 in CBs is dependent on active transcription by Pol II. The concentration of ELL and EAF1 in CBs links the transcriptional elongation activity of ELL to the RNA processing functions previously identified in CBs. Strikingly, CBs are disrupted in MLL-ELL leukemia. EAF1 and p80 coilin are delocalized from CBs in murine MLL-ELL leukemia cells and in HeLa cells transiently transfected with MLL-ELL. Nuclear and cytoplasmic fractionation revealed diminished expression of p80 coilin and EAF1 in the nuclei of MLL-ELL leukemia cells [corrected]. These studies are the first demonstration of a direct role of CB components in leukemogenesis.

ELL-associated factor 2 (EAF2), a functional homolog of EAF1 with alternative ELL binding properties.

The (11;19)(q23;p13.1) translocation in acute leukemia results in the formation of an MLL-ELL fusion protein. ELL is an RNA polymerase II elongation factor that interacts with the recently identified EAF1 protein. To characterize the normal functions of ELL and its aberrant activities when fused to MLL, we isolated a second protein that interacts with ELL named EAF2 for ELL Associated Factor 2. EAF2 is highly homologous to EAF1, with 58% identity and 74% amino acid conservation. Using specific antibodies generated to EAF2, we coimmunoprecipitated ELL and EAF2 from multiple cell lines. Confocal microscopy revealed that endogenous EAF2 and ELL colocalized in a nuclear speckled pattern. Database comparisons with EAF2 identified a region with a high content of serine, aspartic acid, and glutamic acid residues that is conserved with EAF1 and exhibited amino acid similarity with several translocation partner proteins of MLL, including AF4 and ENL. We found that EAF2 and EAF1 both contain transcriptional activation domains within this region. Using retroviral bone marrow transduction, we observed that a heterologous fusion of EAF2 to MLL immortalized hematopoietic progenitor cells. In contrast to EAF1, EAF2 does not bind to the carboxy-terminus of ELL. We identified a protein-protein interaction domain within the amino-terminus of ELL that binds to both EAF1 and EAF2. This amino-terminal interaction domain is disrupted in the formation of the MLL-ELL fusion protein. Thus, MLL-ELL retains an interaction domain for EAF1 but not for EAF2. Taken together, these data suggest that MLL-ELL may disrupt the normal protein-protein interactions of ELL.