ETO and AML1 phosphoproteins are expressed in CD34+ hematopoietic progenitors: implications for t(8;21) leukemogenesis and monitoring residual disease

Abstract

To study acute myelogenous leukemia 1 (AML1) transcription factor, ETO protein, and t(8;21) AML chimeric AML1/ ETO protein in normal hematopoiesis and in leukemia, we raised rabbit antisera to a bacterially expressed polypeptide containing amino acid residues 1 to 220 of ETO and to synthetic peptides extending from residues 528 to 548 of ETO and 32 to 50 of AML1. The latter was selected to have little chance of cross-reactivity with other members of the PEBP2 alpha family. With affinity-purified reagents, we observed immunofluorescent staining for both AML1 and ETO in the nucleus of HEL, K562, and Kasumi- 1 leukemic cell lines, the last from a t(8;21) AML. Biochemical analysis confirmed specificity of the antibodies and the nuclear localization of the antigens, the latter being exclusive for AML1 and primary for ETO. Immunoprecipitations of metabolically labeled 32P- proteins from Kasumi-1 cells show that AML1 and ETO are phosphorylated on serine and threonine. Investigations with normal bone marrow reveal AML1 and ETO are coexpressed in megakaryocytes and that each is expressed in a portion of the approximately 10-microns-diameter cells residing there. Using a CD34+ enriched population mobilized to peripheral blood, we found AML1 and, unexpectedly, ETO present in these cells. Because of this, we conclude that the expression of ETO in hematopoietic cells is not by itself leukemogenic. Also, because ETO would not be exclusively expressed as part of chimeric AML1/ETO in leukemic patients, its presence cannot be used to monitor t(8;21) AML residual disease.

Alternative RUNX1 Promoter Regulation by Wnt/β-Catenin Signaling in Leukemia Cells and Human Hematopoietic Progenitors

“…A), since they resemble normal HSCs (Engelhardt et al, ). As it has been described, low levels of endogenous Runx1 mRNA were observed in isolated CD34 + cells under control conditions (Erickson et al, ). Remarkably, Runx1 transcription in these cells was significantly enhanced in response to short‐term treatment with purified Wnt3a (4 h) and again the effect was mainly observed at the P1 promoter and paralleled by the transcriptional activity of Wnt/β‐catenin target genes (Fig.…”

Decreased expression of transcription factor GATA‐2 in haematopoietic stem cells in patients with aplastic anaemia

“…Among these factors, GATA‐2, SCL and AML1 have been shown to be indispensable for early haematopoiesis at the stem cell level by both in vivo and in vitro study (Tsai et al , 1994; Okuda et al , 1996; Porcher et al , 1996). Furthermore, it has been shown that these factors are expressed in stem cells in the adult haematopoietic system (Mouthon et al , 1993; Erickson et al , 1996). Therefore, it is possible that changes in the expression level of these factors may lead to an aberrant proliferation and differentiation of stem cells, and may be partly responsible for developing stem cell diseases such as aplastic anaemia.…”

Expression of a Knocked-In AML1-ETO Leukemia Gene Inhibits the Establishment of Normal Definitive Hematopoiesis and Directly Generates Dysplastic Hematopoietic Progenitors

“…Although ETO contains a zinc finger motif and appears to be the mammalian homologue of the Drosophila gene nervy, 11 no direct DNAbinding activity has been detected, nor has its function been identified. 12 Transcriptional regulation by AML1 through the enhancer core motif has been shown to be important for the tissue specific expression of a number of hematopoietic specific genes including interleukin-3 (IL-3), 13 granulocyte-macrophage colonystimulating factor (GM-CSF), 14 the receptor for CSF-1 (CSF-1R), 15 myeloperoxidase, 16 and subunits of the T-cell antigen receptor (TCR). 8,17 By creating mice deficient in AML1/CBF␤, we 18 as well as others [19][20][21] have shown that the AML1/CBF␤ transcription factor complex is essential for the establishment of definitive hematopoiesis.…”