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Gene expression changes induced by expression of MN1 deletion mutants in murine bone marrow cells

ABSTRACT: Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia, share the same pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of the malignant disease. We studied the relationship of different protein domains of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal domain of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal domain resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the most N-terminal domain. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active domains. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.

ORGANISM(S): Mus musculus

PROVIDER: GSE46990 | GEO | 2015/01/01

SECONDARY ACCESSION(S): PRJNA203225

REPOSITORIES: GEO

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Project description:Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia, share the same pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of the malignant disease. We studied the relationship of different protein domains of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal domain of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal domain resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the most N-terminal domain. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active domains. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases. C57BL/6J bone marrow cells were harvested from mice treated for 4 days with 150 mg 5-fluorouracil/kg and stimulated for 48 hours in DMEM supplemented with 15% FBS, 10 ng/mL hIL6, 6ng/mL mIL3, and 20ng/mL mSCF. The cells were infected with MN1, MN1Δ1, and MN1Δ7 retroviral constructs by cocultivation with irradiated E86 viral producer cells in the presence of 5μg/mL protamine sulfate (Sigma) for 48 hours and then transplanted into lethally irradiated syngeneic recipient mice. 4 weeks after transplantation, MN1, MN1Δ1, and MN1Δ7 leukemia cells and Gr1+/CD11b+ bone marrow cells were FACS-sorted and analyzed by Affymetrix GeneChip Mouse 430 2.0 (43,000 probes) microarray.

Project description:Myeloid differentiation is blocked in acute myeloid leukemia (AML), but the molecular mechanisms are not well characterized. MN1 is overexpressed in some AML patients and confers resistance to all-trans retinoic acid (ATRA)-induced differentiation. To understand the role of MN1 as a transcriptional regulator in myeloid differentiation, we fused transcriptional activation (VP16) or repression (M33) domains with MN1 and characterized these cells in vivo. Transcriptional activation of MN1 target genes induced myeloproliferative disease with long latency and differentiation potential to mature neutrophils. A large proportion of differentially expressed genes between leukemic MN1 and differentiation-permissive MN1VP16 cells belonged to the immune response pathway like Irf8 and Ccl9. As MN1 is a co-factor of MEIS1 and RARA, we compared chromatin occupancy between MN1, MEIS1 and RARA. Immune response genes that were upregulated in MN1VP16 cells were co-targeted by MN1 and MEIS1, but not RARA, suggesting that myeloid differentiation is blocked through transcriptional repression of shared target genes of MN1 and MEIS1. Constitutive expression of Irf8 or its target gene Ccl9 identified these genes as potent inhibitors of MN1-induced leukemia. Our data show that MN1 prevents activation of the immune response pathway, and suggest that restoration of Irf8 signalling as a novel therapeutic target in AML. C57BL/6J bone marrow cells were harvested from mice treated for 4 days with 150 mg 5-fluorouracil/kg and stimulated for 48 hours in DMEM supplemented with 15% FBS, 10 ng/mL hIL6, 6ng/mL mIL3, and 20ng/mL mSCF. The cells were infected with MN1 or MN1VP16 retroviral constructs by cocultivation with irradiated E86 viral producer cells in the presence of 5μg/mL protamine sulfate (Sigma) for 48 hours and then transplanted into lethally irradiated syngeneic recipient mice. 4 weeks after transplantation MN1, MN1VP16 GFP+ cells and Gr1+/CD11b+ bone marrow cells were FACS-sorted and analyzed by Affymetrix GeneChip Mouse 430 2.0 (43.000 probes) microarrays

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Gene expression changes induced by expression of MN1 deletion mutants in murine bone marrow cells

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