EGFL7 Antagonizes NOTCH Signaling, Stimulates Blast Proliferation and Confers Poor Prognosis in Cytogenetically-Normal Acute Myeloid Leukemia (CN-AML)
Authors: Shen Changxian , Papaioannou Dimitrios , Mcneil Betina , Nicolet Deedra , Karunasiri Malith , Burke Matthew , Nina Zitzer , Behbehani Gregory K. , Ozer Hatice G , Yilmaz Selen , Marcucci Guido , Caligiuri Michael A. , Bloomfield Clara D. , Garzon Ramiro , Dorrance Adrienne 
Published: October 2019

Abstract
Epidermal growth factor-like domain 7 (EGFL7) is a secreted protein and plays an important role in angiogenesis by regulating the growth, proliferation and migration of endothelial cells. Recent studies in solid tumors have shown that EGFL7 is overexpressed and is associated with a more aggressive disease phenotype. Whether EGFL7 plays a similar role in blood cancers such as acute myeloid leukemia (AML) however, has not been previously reported. To investigate the association of EGFL7 expression with outcome in AML, we measured EGFL7 mRNA expression in newly diagnosed older (≥60 years, n=126) CN-AML patients. In these patients, those with high EGFL7 expression were less likely to achieve CR (52% v 76%, P=.009). Patients with high EGFL7 expression status had shorter event-free survival (5-year rates: 6% v 13%, P=.03) and overall survival (5-year rates: 10% v 16%, P=.009) than patients with low EGFL7 expression status.
To validate our clinical data we measured EGFL7 mRNA in primary AML blasts (n=11) compared to normal bone marrow (NBM) (n=5) using RT-PCR, and found a ~2.4 fold-increase in the AML samples, P<0.05. These results were consistent with the protein levels, measured using western blots. Using both ELISA and western blotting, we observed significant increased levels of EGFL7 protein in the medium of cultured primary AML cells (n=3, 31-443 pg/mL) compared to media alone. Importantly, detection of serum EGFL7 levels by ELISA revealed variable but increased levels of EGFL7 in the sera from 3 of 5 (P<0.05) AML patients tested compared with sera from normal healthy controls (n=7). To investigate the functional consequences of EGFL7 stimulation on blast growth we treated primary AML blast cells from three patients with recombinant EGFL7 (rEGFL7) protein and performed colony forming unit (CFU) assays. Treatment with rEGFL7 led to an increase in the number of CFUs (P<0.05) and was associated with increased phosphorylation of AKT. In addition, under starvation conditions, recombinant EGFL7 protein alone promoted the survival and proliferation of MLL-PTD AML cells (n=3) but not normal BM cells (n=3) (P<0.05). To explore the underlying molecular mechanism(s) by which EGFL7 maintains the survival and proliferation of AML cells, we screened for proteins that interact with EGFL7 by using a protein antibody array on human primary AML blasts. This screen revealed that EGFL7 binds to multiple components of key signal transduction pathways important for leukemia, including NOTCH. Previous studies by Lobry C et al., (JExpMed 2013) demonstrated that although NOTCH2 and to a lesser extent, NOTCH1, mRNA was relatively abundant, NOTCH signaling was inhibited in AML blasts. Re-activation of the canonical NOTCH pathway, released a block in differentiation resulting in eradication of the leukemic blasts. Schmidt MH et al., (Nat Cell Biol. 2009) demonstrated that EGFL7 was capable of binding NOTCH receptors and blocking their subsequent activation. Therefore, we hypothesized that one mechanism by which the NOTCH pathway is kept inactive in AML is through autocrine binding of EGFL7 protein to NOTCH receptors. Co-immunoprecipitation assays confirmed binding of EGFL7 to NOTCH2 in both human primary AML cells (n=3, P<0.05) and mouse MLL-PTD AML cells (n=3, P<0.05). Moreover, stimulation of AML blasts with rEGFL7 protein suppressed the cleavage and subsequent activation of NOTCH2, resulting in decreased transcription of NOTCH downstream target genes, HES1 and NRARP (n=3, P<0.05). In summary, we show here that high EGFL7 is associated with worse prognosis in patients with CN-AML, and that EGFL7 is expressed and secreted by the AML cells in an autocrine fashion, promoting their growth in part through antagonizing NOTCH2 activation. Targeting EGFL7 with antibodies to reduce NOTCH binding might represent a novel therapeutic approach to reactivate NOTCH signaling, allowing for blast differentiation and elimination of AML.

Disclosures
No relevant conflicts of interest to declare.