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Development of a high-throughput screening-compatible assay for the discovery of inhibitors of the AF4-AF9 interaction using AlphaScreen technology.
ABSTRACT: 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: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: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: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: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: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:Mixed lineage leukemia (MLL) fusion proteins cause oncogenic transformation of hematopoietic cells by constitutive recruitment of elongation factors to HOX promoters, resulting in overexpression of target genes. The structural basis of transactivation by MLL fusion partners remains undetermined. We show that the ANC1 homology domain (AHD) of AF9, one of the most common MLL translocation partners, is intrinsically disordered and recruits multiple transcription factors through coupled folding and binding. We determined the structure of the AF9 AHD in complex with the elongation factor AF4 and show that aliphatic residues, which are conserved in each of the AF9 binding partners, form an integral part of the hydrophobic core of the complex. Nuclear magnetic resonance relaxation measurements show that AF9 retains significant dynamic behavior which may facilitate exchange between disordered partners. We propose that AF9 functions as a signaling hub that regulates transcription through dynamic recruitment of cofactors in normal hematopoiesis and in acute leukemia.
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: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:MLL is a target of chromosomal translocations in acute leukemias with poor prognosis. The common MLL fusion partner AF9 (MLLT3) can directly bind to AF4, DOT1L, BCOR, and CBX8. To delineate the relevance of BCOR and CBX8 binding to MLL-AF9 for leukemogenesis, here we determine protein structures of AF9 complexes with CBX8 and BCOR, and show that binding of all four partners to AF9 is mutually exclusive. Using the structural analyses, we identify point mutations that selectively disrupt AF9 interactions with BCOR and CBX8. In bone marrow stem/progenitor cells expressing point mutant CBX8 or point mutant MLL-AF9, we show that disruption of direct CBX8/MLL-AF9 binding does not impact in vitro cell proliferation, whereas loss of direct BCOR/MLL-AF9 binding causes partial differentiation and increased proliferation. Strikingly, loss of MLL-AF9/BCOR binding abrogated its leukemogenic potential in a mouse model. The MLL-AF9 mutant deficient for BCOR binding reduces the expression of the EYA1 phosphatase and the protein level of c-Myc. Reduction in BCOR binding to MLL-AF9 alters a MYC-driven gene expression program, as well as altering expression of SIX-regulated genes, likely contributing to the observed reduction in the leukemia-initiating cell population.
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.