A murine Mll-AF4 knock-in model results in lymphoid and myeloid deregulation and hematologic malignancy.
ABSTRACT: The 2 most frequent human MLL hematopoietic malignancies involve either AF4 or AF9 as fusion partners; each has distinct biology but the role of the fusion partner is not clear. We produced Mll-AF4 knock-in (KI) mice by homologous recombination in embryonic stem cells and compared them with Mll-AF9 KI mice. Young Mll-AF4 mice had lymphoid and myeloid deregulation manifest by increased lymphoid and myeloid cells in hematopoietic organs. In vitro, bone marrow cells from young mice formed unique mixed pro-B lymphoid (B220(+)CD19(+)CD43(+)sIgM(-), PAX5(+), TdT(+), IgH rearranged)/myeloid (CD11b/Mac1(+), c-fms(+), lysozyme(+)) colonies when grown in IL-7- and Flt3 ligand-containing media. Mixed lymphoid/myeloid hyperplasia and hematologic malignancies (most frequently B-cell lymphomas) developed in Mll-AF4 mice after prolonged latency; long latency to malignancy indicates that Mll-AF4-induced lymphoid/myeloid deregulation alone is insufficient to produce malignancy. In contrast, young Mll-AF9 mice had predominately myeloid deregulation in vivo and in vitro and developed myeloid malignancies. The early onset of distinct mixed lymphoid/myeloid lineage deregulation in Mll-AF4 mice shows evidence for both "instructive" and "noninstructive" roles for AF4 and AF9 as partners in MLL fusion genes. The molecular basis for "instruction" and secondary cooperating mutations can now be studied in our Mll-AF4 model.
Project description: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.
Project description:Transcriptome analysis by RNAseq of leukemia model promoted by MLL-Af4 or MLL-AF9 fusion proteins. We find each fusion protein promotes a specific gene signature correlating to those identified in patients Overall design: Human CD34+ hematopoietic stem and progenitor cells were transduced with retrovirus expressing MLL-Af4 or MLL-AF9. Transduced cells were transplanted into immunodeficient mice to induce lymphoid leukemia or placed in myeloid in vitro culture. CD19+ lymphoid leukemia cells (3 AF9, 6 Af4), control health CD19+CD34+ proB cells (n=3) and 4 pairs of Af4 and AF9 CD33+CD19- myeloid culture cells were collected for RNA-seq
Project description:MLL, involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia, has >50 known partner genes with which it is able to form in-frame fusions. Characterizing important downstream target genes of MLL and of MLL fusion proteins may provide rational therapeutic strategies for the treatment of MLL-associated leukemia. We explored downstream target genes of the most prevalent MLL fusion protein, MLL-AF4. To this end, we developed inducible MLL-AF4 fusion cell lines in different backgrounds. Overexpression of MLL-AF4 does not lead to increased proliferation in either cell line, but rather, cell growth was slowed compared with similar cell lines inducibly expressing truncated MLL. We found that in the MLL-AF4-induced cell lines, the expression of the cyclin-dependent kinase inhibitor gene CDKN1B was dramatically changed at both the RNA and protein (p27kip1) levels. In contrast, the expression levels of CDKN1A (p21) and CDKN2A (p16) were unchanged. To explore whether CDKN1B might be a direct target of MLL and of MLL-AF4, we used chromatin immunoprecipitation (ChIP) assays and luciferase reporter gene assays. MLL-AF4 binds to the CDKN1B promoter in vivo and regulates CDKN1B promoter activity. Further, we confirmed CDKN1B promoter binding by ChIP in MLL-AF4 as well as in MLL-AF9 leukemia cell lines. Our results suggest that CDKN1B is a downstream target of MLL and of MLL-AF4, and that, depending on the background cell type, MLL-AF4 inhibits or activates CDKN1B expression. This finding may have implications in terms of leukemia stem cell resistance to chemotherapy in MLL-AF4 leukemias.
Project description:All-trans retinoic acid (ATRA) is well established as differentiation therapy for acute promyelocytic leukemia (APL) in which the PML-RAR? (promyelocytic leukemia-retinoic acid receptor ?) fusion protein causes blockade of the retinoic acid (RA) pathway; however, in types of acute myeloid leukemia (AML) other than APL, the mechanism of RA pathway inactivation is not fully understood. This study revealed the potential mechanism of high ATRA sensitivity of mixed-lineage leukemia (MLL)-AF9-positive AML compared with MLL-AF4/5q31-positive AML. Treatment with ATRA induced significant myeloid differentiation accompanied by upregulation of RAR?, C/EBP?, C/EBP? and PU.1 in MLL-AF9-positive but not in MLL-AF4/5q31-positive cells. Combining ATRA with cytarabine had a synergistic antileukemic effect in MLL-AF9-positive cells in vitro. The level of dimethyl histone H3 lysine 4 (H3K4me2) in the RAR? gene-promoter region, PU.1 upstream regulatory region (URE) and RUNX1+24/+25 intronic enhancer was higher in MLL-AF9-positive cells than in MLL-AF4-positive cells, and inhibiting lysine-specific demethylase 1, which acts as a histone demethylase inhibitor, reactivated ATRA sensitivity in MLL-AF4-positive cells. These findings suggest that the level of H3K4me2 in the RAR? gene-promoter region, PU.1 URE and RUNX1 intronic enhancer is determined by the MLL-fusion partner. Our findings provide insight into the mechanisms of ATRA sensitivity in AML and novel treatment strategies for ATRA-resistant AML.
Project description:Patients with a t(9;11) translocation (MLL-AF9) develop acute myeloid leukemia (AML), and while in mice the expression of this fusion oncogene also results in the development of myeloid leukemia, it is with long latency. To identify mutations that cooperate with Mll-AF9, we infected neonatal wild-type (WT) or Mll-AF9 mice with a murine leukemia virus (MuLV). MuLV-infected Mll-AF9 mice succumbed to disease significantly faster than controls presenting predominantly with myeloid leukemia while infected WT animals developed predominantly lymphoid leukemia. We identified 88 candidate cancer genes near common sites of proviral insertion. Analysis of transcript levels revealed significantly elevated expression of Mn1, and a trend toward increased expression of Bcl11a and Fosb in Mll-AF9 murine leukemia samples with proviral insertions proximal to these genes. Accordingly, FOSB and BCL11A were also overexpressed in human AML harboring MLL gene translocations. FOSB was revealed to be essential for growth in mouse and human myeloid leukemia cells using shRNA lentiviral vectors in vitro. Importantly, MN1 cooperated with Mll-AF9 in leukemogenesis in an in vivo BM viral transduction and transplantation assay. Together, our data identified genes that define transcription factor networks and important genetic pathways acting during progression of leukemia induced by MLL fusion oncogenes.
Project description:Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34(+)CD38(+)CD19(+) and CD34(-)CD19(+) cells initiated leukemia, and in MLL-AF9 patients, CD34(-)CD19(+) cells were LICs. In MLL-ENL patients, either CD34(+) or CD34(-) cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34(+)CD38(-)CD19(-)CD33(-) cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34(+)CD38(-)CD19(-)CD33(-) cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4(+) single positive (SP), CD8(+) SP, and CD4(+)CD8(+) double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34(+)CD38(+) and CD34(-) LICs but not in CD34(+)CD38(-)CD19(-)CD33(-) HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia.
Project description:Rearrangements of the mixed-lineage leukemia (MLL) gene occur predominately in pediatric leukemia cases and are generally predictors of a poor prognosis. These chromosomal rearrangements result in fusion of the protein MLL to one of more than 60 protein partners. MLL fusions are potent inducers of leukemia through activation of oncogene expression; therefore, targeting this transcriptional activation function may arrest MLL-rearranged (MLL-R) leukemia. Leukemic cell lines harboring the most common fusion protein, MLL-AF4, require the direct interaction of AF4 with the transcription factor AF9 to survive and self-renew; disrupting this interaction with a cell-penetrating AF4-derived peptide results in cell death, suggesting that the AF4-AF9 interaction could be a viable target for a novel MLL-R leukemia therapy. Here we describe the use of AlphaScreen technology to develop a high-throughput screening (HTS) assay to detect nonpeptidic inhibitors of AF4-AF9 binding. The assay is economical, requiring only low nanomolar concentrations of biotinylated AF4-derived peptide and FLAG-tagged AF9 in low-volume 384-well plates. A Z'-factor of 0.71 and a signal-to-background ratio of 21.3 showed the assay to be robust, and sensitivity to inhibition was demonstrated with competing AF4-derived peptides. Two pilot screens comprising 5,680 compounds served as validation for HTS at Nemours and the Broad Institute. Assay artifacts were excluded using a counterscreen comprising a biotinylated FLAG peptide. This is the first reported HTS-compatible assay to identify compounds that inhibit a key binding interaction of an MLL fusion partner, and the results presented here demonstrate suitability for screening large chemical libraries in high-density, low-volume plate formats.
Project description:A number of acute leukemias arise from fusion of the mixed lineage leukemia 1 protein (MLL) N terminus to a variety of fusion partners that have been reported to reside in one or more poorly defined complexes linked to transcription elongation through interactions with the histone H3-K79 methyltransferase DOT1 and positive transcription elongation factor b (P-TEFb). Here we first identify natural complexes (purified through fusion partners AF9, AF4, and ELL) with overlapping components, different elongation activities, and different cofactor associations that suggest dynamic interactions. Then, through reconstitution of defined, functionally active minimal complexes, we identify stable subcomplexes that, through newly defined protein-protein interactions, form distinct higher order complexes. These definitive analyses show, for example, that (i) through direct interactions with AF9 and cyclinT1, family members AF4 and AFF4 independently mediate association of P-TEFb with AF9, (ii) P-TEFb, through direct interactions, provides the link for association of ELL and ELL-associated factors 1 and 2 (EAF1 and EAF2) with AF4, and (iii) in the absence of other factors, DOT1 forms a stable complex with AF9 and does not interact with AF9•AF4•P-TEFb complexes. Finally, we show the importance of defined higher order complex formation in MLL-AF9-mediated transcriptional up-regulation and cell immortalization potential in vivo. Thus, our study provides direct mechanistic insight into the role of fusion partners in MLL fusion-mediated leukemogenesis.
Project description: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:Erythropoietin-producing hepatoma-amplified sequence (Eph) receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, function as a unique signaling system triggered by cell-to-cell interaction and have been shown to mediate neurodevelopmental processes. In addition, recent studies showed deregulation of some of Eph/ephrin genes in human malignancies, suggesting the involvement of this signaling pathway in tumorigenesis. The ALL1 (also termed MLL) gene on human chromosome 11q23 was isolated by virtue of its involvement in recurrent chromosome translocations associated with acute leukemias with poor prognosis. The translocations fuse ALL1 to any of >50 partner genes and result in production of chimeric proteins composed of the ALL1 N terminus and the C terminus of the partner protein. The most common translocations in ALL1-associated leukemias are t(4;11) and t(9;11), which generate ALL1/AF4 and ALL1/AF9 fusion protein, respectively. In the present study, we sought to determine whether ALL1 fusion proteins are involved in regulation of Eph/ephrin genes. Screening of K562 cells producing recombinant ALL1/AF4 or ALL1/AF9 fusion protein revealed transcriptional up-regulation of the EphA7. Consistent with this finding, siRNA-mediated suppression of ALL1/AF4 in SEMK2 cells carrying the t(4;11) chromosome translocation resulted in down-regulation of EphA7. ChIP analysis demonstrated the occupancy of tagged ALL1 fusion proteins on the EphA7 promoter, pointing to EphA7 as a direct target of the formers. Further studies demonstrate that EphA7 up-regulation is accompanied by ERK phosphorylation. Finally, we show apoptotic cell death, specific for leukemic cells carrying the t(4;11) chromosome translocation, after treatment of the cells with an ERK phosphorylation blocker.