The Role of VEGFA in Malignant Progression in AML

Abstract Vascular Endothelial Growth Factor (VEGFA) at time of diagnosis is an independent prognostic factor for poor treatment outcome in (pediatric) acute myeloid leukemia (AML). Inhibition of VEGFA by Bevacizumab, a recombinant humanized monoclonal antibody, preceded by chemotherapy yields a favorable CR rate and duration in adults with refractory AML (Karp et al., 2004). VEGFA is a powerful stimulator of angiogenesis; VEGFA binds to the tyrosine kinase receptors VEGFR1 and VEGFR2 on endothelial cells, resulting in endothelial cell proliferation. Moreover, binding of VEGFA to VEGF receptors on AML cells in vitro promotes leukemic cell survival via activation of signaling pathways such as RAS/Raf/MEK/ERK and PI3K/AKT pathway. In our study a model was generated to investigate the effect of VEGFA in malignant progression in AML. An HL-60 AML cell line was transduced with VEGFA (the splice variant VEGFA165) or a control vector using a retroviral construct. With RT-PCR we found a threefold induction of VEGFA in VEGFA165 transduced cells. No difference in growth rate and drug sensitivity was found between the HL-60 VEGFA165 cells and HL-60 control cells in vitro. Thus, VEGFA165 overexpression did not result in growth benefit in vitro. To evaluate the in vivo effects of VEGFA165 overexpression, we injected 10 × 106 HL-60 VEGFA165 cells or HL-60 control cells s.c. into NOD/SCID mice (n=14). We observed that overexpression of VEGFA165 increased tumor weight (median weight: HL-60 VEGFA165 tumors 995 mg, range 670–1344; HL-60 control tumors 464 mg, range 413–646; p=0.001). So, the effects of AML overexpressed VEGFA165 are detectable in combination with its environment. Using gene expression profiles (Affymetrix) we found differentially activated signaling pathways, e.g. the PI3K/AKT (p<0.001), MAPK (p<0.001), Jak-STAT (p<0.001) and VEGF-pathway (p<0.001), as well as pathways involved in cell interaction. With GeneTrail (a web-based application that scores a sorted list of genes with respect to their enrichment of functional categories) and the transcriptional system regulator approach (De Jonge et al., 2008) we could demonstrate that the Jak-STAT pathway (p=0.02) as well as the apoptosis pathway (p=0.04) was more active in the VEGFA165 HL-60 tumors, whereas the cytokine-cytokine receptor interaction was more active in de HL-60 control tumors (p=0.02). In addition, we found differential expression of the process angiogenesis (p=0.002). Cell proliferation within the tumors was verified by staining for Ki67; the HL-60 VEGFA165 tumor cells showed more proliferation than the HL-60 control tumor cells, as a netto result of multiple signaling pathway activation (p=0.02). In conclusion, overexpression of VEGFA165 did not have a growth benefit in vitro, whereas in vivo an increase in tumor volume was seen when VEGFA165 was overexpressed. VEGFA related pro-angiogenic effects are found in the AML tumor cells as well as enhanced signaling and proliferation in AML tumor cells in vivo. Therefore, the interaction of VEGFA165 with its environment plays a critical role in the malignant progression in AML. New design drugs related to VEGF/VEGFR interaction need to be tested in context of a tumor in its environment.