The Angiogenic Factor Angiopoietin-1 Is Regulated by the Acute Myeloid Leukemia Fusion Protein AML1/ETO

The chromosomal rearrangement t(8;21)(q22;q22) encodes the fusion gene AML1/ETO, which is the most common translocation in acute myeloid leukemia (AML). It has an incidence of approximately 15% and a favourable prognosis in comparison to other AML subtypes. Dysregulated angiogenesis in the bone marrow niche environment is predicted to have an important role in leukemia pathogenesis, and several factors have been implicated in this process. Angiopoietin-1 (ANGPT1) is a cytokine involved in hematopoietic stem cell quiescence and regulation of microvessel density within the bone marrow, as well as having a role in transendothelial migration. Moreover, ANGPT1 is upregulated in leukemic blast cells from AML patients. In this study we investigated the role of AML1/ETO as a regulator of ANGPT1 expression, as well as functional implications of ANGPT1 in AML1/ETO-positive AML.

In order to investigate putative AML1/ETO-dependent regulation of ANGPT1, we performed gain-of-function studies using lentiviral gene transfer to ectopically express AML1/ETO in HL-60 and U937 AML cell lines. We also depleted AML1/ETO in the t(8;21)-positive AML cell line Kasumi-1 using fusion gene specific siRNA. Additionally, the functional role of ANGPT1 was studied using targeted RNAi in Kasumi-1 cells. Transcript expression of AML1/ETO and ANGPT1 was analysed by quantitative Real-Time PCR (qRT-PCR) and AML1/ETO protein expression was quantified by western blotting. Angiopoietin-1 protein secretion was determined using enzyme-linked immunosorbent assay (ELISA). We also utilised Matrigel transwell assays to test the effect of ANGPT1 downregulation on the invasive and migratory properties of Kasumi-1.

In HL-60 and U937 transduced with AML1/ETO, we observed an up to 280 fold increase in ANGPT1 mRNA transcript levels as measured by qRT-PCR, which correlated with an increase in secreted Angiopoietin-1 protein. Conversely, siRNA-mediated AML1/ETO depletion in Kasumi-1 cells significantly decreased ANGPT1 transcript and protein levels after a single electroporation. After three serial electroporations with siRNA, AML1/ETO transcript levels were reduced by 85%, with a concomitant decline in ANGPT1 transcript (>99%) and secreted protein. Preliminary data suggest siRNA targeting of ANGPT1 in Kasumi-1 decreases the invasive ability of these cells, causing a ≥50% reduction in invasion when compared to controls.

We have identified a strong correlation between AML1/ETO and ANGPT1 expression, whereby a reduction of AML1/ETO results in a substantial reduction of ANGPT1. Similarly, the introduction of AML1/ETO into myeloid cell lines results in a large upregulation of ANGPT1. Preliminary evidence suggests that a reduction of ANGPT1 reduces the invasive and migratory potential of Kasumi-1. This could have major functional consequences in the bone marrow niche with regards to understanding the AML stem cell and its interaction with the niche environment as well as providing insight into how leukemic cells in the circulation might interact with the vasculature.

No relevant conflicts of interest to declare.