Subject(s)
Core Binding Factor Alpha 2 Subunit/physiology , DNA-Binding Proteins/genetics , Leukemia, Myeloid/pathology , Oncogene Proteins, Fusion , Proto-Oncogene Proteins/genetics , RUNX1 Translocation Partner 1 Protein/physiology , Transcription Factors/genetics , Age Factors , Animals , Leukemia, Myeloid/genetics , Leukemia, Myeloid/metabolism , Mice , Mice, Knockout , Translocation, GeneticABSTRACT
Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect normal hematopoiesis. The analysis of human AMLs has mostly been performed using end-point materials, such as cell lines and patient derived AMLs that also carry additional contributing mutations. The molecular effects of a single oncogenic hit, such as expression of the AML associated oncoprotein AML1-ETO on hematopoietic development and transformation into a (pre-) leukemic state still needs further investigation. Here we describe the development and characterization of an induced pluripotent stem cell (iPSC) system that allows in vitro differentiation towards different mature myeloid cell types such as monocytes and granulocytes. During in vitro differentiation we expressed the AML1-ETO fusion protein and examined the effects of the oncoprotein on differentiation and the underlying alterations in the gene program at 8 different time points. Our analysis revealed that AML1-ETO as a single oncogenic hit in a non-mutated background blocks granulocytic differentiation, deregulates the gene program via altering the acetylome of the differentiating granulocytic cells, and induces t(8;21) AML associated leukemic characteristics. Together, these results reveal that inducible oncogene expression during in vitro differentiation of iPS cells provides a valuable platform for analysis of aberrant regulation in disease.
Subject(s)
Cell Differentiation/genetics , Cell Transformation, Neoplastic/genetics , Core Binding Factor Alpha 2 Subunit/physiology , Granulocytes/physiology , Induced Pluripotent Stem Cells/physiology , Oncogene Proteins, Fusion/physiology , RUNX1 Translocation Partner 1 Protein/physiology , Transcriptome , Cell Proliferation/genetics , Cells, Cultured , Core Binding Factor Alpha 2 Subunit/genetics , Granulocytes/metabolism , Humans , Leukemia, Myeloid, Acute/genetics , Leukopoiesis/genetics , Monocytes/physiology , Myelopoiesis/genetics , Oncogene Proteins, Fusion/genetics , Oncogenes/physiology , RUNX1 Translocation Partner 1 Protein/genetics , Transcriptome/genetics , TransfectionSubject(s)
Core Binding Factor Alpha 2 Subunit/physiology , Leukemia, Myeloid, Acute/pathology , Oncogene Proteins, Fusion/physiology , Protein Tyrosine Phosphatases/metabolism , RUNX1 Translocation Partner 1 Protein/physiology , Animals , Cell Line, Tumor , Humans , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/genetics , Mice , Protein Tyrosine Phosphatases/genetics , Survival RateABSTRACT
The AML1/ETO fusion protein found in acute myeloid leukemias functions as a transcriptional regulator by recruiting co-repressor complexes to its DNA binding site. In order to extend the understanding of its role in preleukemia, we expressed AML1/ETO in a murine immortalized pluripotent hematopoietic stem/progenitor cell line, EML C1, and found that genes involved in functions such as cell-to-cell adhesion and cell motility were among the most significantly regulated as determined by RNA sequencing. In functional assays, AML1/ETO-expressing cells showed a decrease in adhesion to stromal cells, an increase of cell migration rate in vitro, and displayed an impairment in homing and engraftment in vivo upon transplantation into recipient mice. Our results suggest that AML1/ETO expression determines a more mobile and less adherent phenotype in preleukemic cells, therefore altering the interaction with the hematopoietic niche, potentially leading to the migration across the bone marrow barrier and to disease progression.
Subject(s)
Hematopoietic Stem Cells/physiology , Neoplastic Stem Cells/physiology , Oncogene Proteins, Fusion/physiology , Animals , Cell Adhesion/genetics , Cell Adhesion/physiology , Cell Line, Tumor , Cell Movement/genetics , Cell Movement/physiology , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/physiology , Female , Hematopoietic Stem Cells/pathology , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/physiopathology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Neoplastic Stem Cells/pathology , Oncogene Proteins, Fusion/genetics , RUNX1 Translocation Partner 1 Protein/genetics , RUNX1 Translocation Partner 1 Protein/physiology , Stem Cell Niche/genetics , Stem Cell Niche/physiology , Tumor Microenvironment/geneticsABSTRACT
Chromosomal translocation 8;21 is found in 40% of the FAB M2 subtype of acute myeloid leukemia (AML). The resultant in-frame fusion protein AML1-ETO (AE) acts as an initiating oncogene for leukemia development. AE immortalizes human CD34(+) cord blood cells in long-term culture. We assessed the transforming properties of the alternatively spliced AE isoform AE9a (or alternative splicing at exon 9), which is fully transforming in a murine retroviral model, in human cord blood cells. Full activity was realized only upon increased fusion protein expression. This effect was recapitulated in the AE9a murine AML model. Cotransduction of AE and AE9a resulted in a strong selective pressure for AE-expressing cells. In the context of AE, AE9a did not show selection for increased expression, affirming observations of human t(8;21) patient samples where full-length AE is the dominant protein detected. Mechanistically, AE9a showed defective transcriptional regulation of AE target genes that was partially corrected at high expression. Together, these results bring an additional perspective to our understanding of AE function and highlight the contribution of oncogene expression level in t(8;21) experimental models.
