AML1/RUNX1, One of the Most Common Targets of Aberration in Acute Myeloid Leukemia as a Transcriptional Regulator of Vascular Endothelial Growth Factor (VEGFA).
Authors: Elst Arja ter , Ma Bin , Scherpen Frank , Wierenga Bart-Jan A.T. , Schuringa Jan Jacob , Douwes Jenny , Kamps Willem A. , de Bont Evelina S. 
Published: April 2020

Cellular VEGFA is an independent adverse prognostic factor related to worse outcome in AML and is significantly upregulated in leukemic blasts (de bont ES. et al., 2002). Transcriptional activation of the VEGFA promoter represents the core mechanism through which expression of VEGFA can be regulated, but is still not identified in AML (Carmeliet P. and Jaine RK., 2000; Kolch W. et al., 1995; Yancoupolus GD. et al., 2000). One of the most common targets of aberrations in acute leukemia is Runt domain transcription factor (AML1/RUNX1), due to point mutations (Roumier C. et al., 2003) or disruption of chromosome 21, which occurs in about 10%-15% of all de novo AML patients. In the current study, functional 5′ deletion analysis of the VEGFA promoter was performed to identify VEGFA promoter regions involved in VEGFA transcriptional regulation in AML. Loss of the region spanning −2274/−507 resulted in a significant increase of promoter activity in two AML cell lines, whereas the promoter activity dropped after deletion of the promoter region −286 to −52. Computer-assisted sequence analysis of the −2247/−507 fragment of the VEGFA promoter, revealed three perfect AML1/RUNX1-binding sites in the first three deletion regions. The loss of each AML1/RUNX1-binding site corresponded to an (further) increase in VEGFA promoter activity. siRNA mediated AML1/RUNX1 depletion caused a 24% to 36% increase in VEGFA mRNA expression in two different AML cell lines (HL-60 and TF-1), whereas VEGFA promoter activity was two-fold increased in HL-60 cells treated with AML1/RUNX1 siRNA compared to the non-silencing control siRNA. In addition, mutation of all three AML1/RUNX1 sites resulted in a 22 fold increase of VEGFA promoter activity in HL-60 cells. VEGFA mRNA expression was found to be higher in AML patients with a t(8;21) translocation compared to patients without this translocation. To investigate whether blocking of the fusion protein AML1-ETO, generated by the t(8;21) translocation, would have an effect on VEGFA mRNA we have used a siRNA against AML-ETO in Kasumi-1. siRNA mediated depletion of AML1-ETO caused a 40% decrease in VEGFA mRNA expression. AML-ETO has the potential to interact with AML1/RUNX1 co-factors such as histone deacetylase (HDAC) which results in a dominant negative effect on AML1/RUNX1 transcriptional regulation. To test whether AML1/RUNX1 transcription repression of VEGFA is histone deacetylase (HDAC) dependent HL-60 cells were treated with 200 nM Trichostatin A (TSA), a potent inhibitor of HDAC, this resulted in an 75% increase of luciferase activity after 24h. In contrast, TSA treatment of Kasumi-1 cells containing the AML-ETO fusion protein had no effect on VEGFA expression. These results indicate that VEGFA is transcriptionally regulated by AML1/RUNX1, one of the most common targets of aberrations in AML, through three AML1/RUNX1 repressor elements located in the promoter of VEGFA. Furthermore, prognostically unfavorable high VEGFA expression in AML is caused by a dominant negative effect of the fusion protein AML-ETO on AML1/RUNX1 mediated repression of VEGFA transcription. These results underscore the importance of AML1/RUNX1 aberrations in AML development and progression and better understanding the molecular basis for aberrant AML/RUNX1 signaling pathway may help design more effective treatment strategies.