EGFL7 Antagonizes NOTCH Signaling and Represents a Novel Therapeutic Target in Acute Myeloid Leukemia (AML)
Authors: Bill Marius , Pathmanathan Aparna , Karunasiri Malith , Shen Changxian , Burke Matthew , Ranganathan Parvathi , Papaioannou Dimitrios , Zitzer Nina C. , Snyder Katiri , LaRocco Allison , Walker Allison , Brannan Zachary , Nalin Ansel P. , Freud Aharon G. , Dikov Mikhail , Zhang Xiaoli , Bloomfield Clara D. , Garzon Ramiro , Dorrance Adrienne M. 
Published: November 2019

Epidermal growth factor-like domain 7 (EGFL7) is a secreted protein. We previously found that EGFL7 is up-regulated in primary AML blasts and that a high mRNA expression correlates with shorter event-free and overall survival in AML patients (Papaioannou et al. PNAS 2017;114(23)). However, the underlying mechanism by which EGFL7 promotes leukemogenesis is largely unknown.
To identify EGFL7 binding partners, we took an unbiased high-throughput approach by using an antibody interaction-array to measure the ability of EGFL7 to bind directly ~400 proteins expressed by primary AML blasts. Using this strategy, we found in cell lysates of 3 AML patients that EGFL7 binds several signaling proteins important for normal and malignant hematopoiesis including NOTCH (in all patients P<0.001 compared to control). The direct interaction between EGFL7 and NOTCH1 as well as NOTCH2 receptor was confirmed in co-immunoprecipitation assays in THP1 and primary patient cells.
Next, we stimulated AML blasts from 3 patients with recombinant EGFL7 (rEGFL7) and found that it reduced NOTCH intracellular domain (NICD1/2, Figure 1A), indicating that EGFL7 inhibits NOTCH activation. In line with this, we found that stimulation of AML patients samples' with rEGFL7 resulted in a decreased expression of its well-known downstream target gene HES1 (Figure 1B; in all patients P<0.05 compared to unstimulated (unstim) cells).
On the other hand, treatment with an anti-EGFL7 blocking antibody (AB) caused increased levels of NICD1/2 (Figure 1C) and expression of HES1 (Figure 1D, in all patients P<0.05 compared to cells treated with IgG) indicating a reactivation of NOTCH signaling. Moreover, the treatment of THP1 cells with the anti-EGFL7 blocking AB resulted in increased apoptosis (1.9 fold increase compared to IgG control, P<0.05) and differentiation (14.9 fold increase in CD11B expression compared to IgG control, P<0.05; 5.6 fold increase in CD14 expression compared to IgG control, P<0.05).
To determine whether blocking EGFL7 could provide a new targeted therapy for patients with AML, we treated 3 independent AML models (i.e. an AML cell line based xenotransplant model (n=4 mice per group), a primary murine AML model (n=7 mice per group), and a patient derived xenotransplant model (n=4 mice per group)) with anti-EGFL7 or IgG1 control to determine whether anti-EGFL7 could prolong survival in vivo. In all models, we demonstrated that in vivo treatment with anti-EGFL7 results in prolonged overall survival (cell line model: P<0.01; primary murine AML model: P<0.01; for the patient derived xenotransplant model: P<0.05).
In conclusion, our data demonstrate that EGFL7 contributes to NOTCH silencing in AML by antagonizing canonical NOTCH ligand binding. Reactivation of NOTCH signaling in vivo using anti-EGFL7 results in prolonged survival of leukemic mice, supporting EGFL7 might be a novel therapeutic target in AML.
Figure 1 A AML patient blasts were cultured in the presence or absence (Unstim) of rEGFL7. Total proteins were extracted for immunoblotting with β-ACTIN as loading control. B Total RNA was extracted for quantitative real time (qRT-PCR) analysis of HES1 mRNA normalized to β-ACTIN control (*P<0.05, **P<0.01, ***P<0.001) stimulated with +rEGFL7 vs Unstim. C Patient blasts were cultured in presence of 100 μg/ml of normal IgG or 100 μg/ml anti-EGFL7 antibody (@EGFL7) for 4 hours. Total protein was extracted for immunoblotting of NICD with b-ACTIN as loading control. D Patient blasts were cultured with 100 μg/ml of normal IgG or anti-EGFL7 antibody for 2 hours and 10 hours. Total RNA was extracted for qRT-PCR analysis of HES1 mRNA with b-ACTIN as internal control. *P<0.05, ***P<0.001 vs IgG control. E Treatment with anti-EGFL7 blocking antibody prolonged survival compared to IgG1 controls in 3 independent mouse models.

Disclosures
No relevant conflicts of interest to declare.