Stage specific over-expression of the dominant negative Ikaros 6 reveals distinct role of Ikaros throughout human B-cell differentiation
ABSTRACT Ikaros is a transcription factor that acts both as an activator and as an inhibitor of gene expression in several hematopoietic lineages. Ikaros functions in hematopoiesis have mostly been studied in mice, and are notably crucial for lymphopoiesis. Deregulation of Ikaros expression was evidenced in several leukemia subtypes, including pre-B-ALL. Here, we studied the role of Ikaros in human B lymphoid differentiation through xeno-transplantation of genetically modified cord blood (CB) human hematopoietic progenitor cells (HPC) in NOD/SCID mice. We used lentiviral vectors to force expression of Ikaros 6 (Ik6), a known dominant negative (DN) protein that interferes with normal Ikaros and structurally related proteins in HPC and their progeny. Two types of vectors were used: a vector containing the EF1alpha promoter which produces strong gene expression in all hematopoietic lineages, and a recently validated B-specific vector containing an enhanced CD19 derived promoter that strongly favors expression in the B-cell lineage. Ik6 transduction of CB CD34(+) cells with these vectors produced distinct consequences in the B-cell differentiation profiles of xenografted human cells. While the ubiquitous vector favored a specific block at the early pro-B/pre-B stage of differentiation, with an increase in Lambda Like transcript expression in the bone marrow (BM), B-specific Ik6 expression provoked a global decrease in the CD19(+) cell population in both BM and spleen, associated with a decrease in IgM+ immature B-cells in the spleen. We conclude that Ikaros proteins are active throughout human B-cell differentiation, before and after CD19 appearance.
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ABSTRACT: Disrupted IKAROS activity is a recurrent feature of some human leukemias, but effects on normal human hematopoietic cells are largely unknown. Here, we used lentivirally mediated expression of a dominant-negative isoform of IKAROS (IK6) to block normal IKAROS activity in primitive human cord blood cells and their progeny. This produced a marked (10-fold) increase in serially transplantable multipotent IK6+ cells as well as increased outputs of normally differentiating B cells and granulocytes in transplanted immunodeficient mice, without producing leukemia. Accompanying T/natural killer (NK) cell outputs were unaltered, and erythroid and platelet production was reduced. Mechanistically, IK6 specifically increased human granulopoietic progenitor sensitivity to two growth factors and activated CREB and its targets (c-FOS and Cyclin B1). In more primitive human cells, IK6 prematurely initiated a B cell transcriptional program without affecting the hematopoietic stem cell-associated gene expression profile. Some of these effects were species specific, thus identifying novel roles of IKAROS in regulating normal human hematopoietic cells.Stem Cell Reports 10/2014; 3(11). DOI:10.1016/j.stemcr.2014.09.006
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ABSTRACT: Over the past two decades, hematologic malignancies have been extensively evaluated due to the introduction of powerful technologies, such as conventional karyotyping, FISH analysis, gene and microRNA expression profiling, array comparative genomic hybridization and SNP arrays, and next-generation sequencing (including whole-exome sequencing and RNA-seq). These analyses have allowed for the refinement of the mechanisms underlying the leukemic transformation in several oncohematologic disorders and, more importantly, they have permitted the definition of novel prognostic algorithms aimed at stratifying patients at the onset of disease and, consequently, treating them in the most appropriate manner. Furthermore, the identification of specific molecular markers is opening the door to targeted and personalized medicine. The most important findings on novel acquisitions in the context of acute lymphoblastic leukemia of both B and T lineage and de novo acute myeloid leukemia are described in this review. © 2014 S. Karger AG, Basel.Medical Principles and Practice 06/2014; 23(6). DOI:10.1159/000362793 · 1.11 Impact Factor