No abstract

Purpose: EGF-like domain 7 (EGFL7) is a secreted protein and recently has been shown to play an important role in acute myeloid leukemia (AML); however, the underlying mechanism by which EGFL7 promotes leukemogenesis is largely unknown.Experimental Design: Using an antibody interaction array, we measured the ability of EGFL7 to bind directly approximately 400 proteins expressed by primary AML blasts. Primary patient samples were stimulated in vitro with recombinant EGFL7 (rEGFL7) or anti-EGFL7 blocking antibody to assess alterations in downstream signaling and the ability to effect blast differentiation and survival. We treated three independent AML models with anti-EGFL7 or IgG1 control to determine whether anti-EGFL7 could prolong survival in vivo.Results: We found EGFL7 significantly binds several signaling proteins important for normal and malignant hematopoiesis including NOTCH. Stimulation of AML blasts with rEGFL7 reduced NOTCH intracellular domain and NOTCH target gene expression while treatment with an anti-EGFL7 blocking antibody resulted in reactivation of NOTCH signaling, increased differentiation, and apoptosis. Competitive ligand-binding assays showed rEGFL7 inhibits DELTA-like (DLL) 4-mediated NOTCH activation while anti-EGFL7 combined with DLL4 significantly increased NOTCH activation and induced apoptosis. Using three different AML mouse models, we demonstrated that in vivo treatment with anti-EGFL7 alone results in increased survival.Conclusions: 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 the use of EGFL7 as a novel therapeutic target in AML.

<div>AbstractPurpose:<p>EGF-like domain 7 (EGFL7) is a secreted protein and recently has been shown to play an important role in acute myeloid leukemia (AML); however, the underlying mechanism by which EGFL7 promotes leukemogenesis is largely unknown.</p>Experimental Design:<p>Using an antibody interaction array, we measured the ability of EGFL7 to bind directly approximately 400 proteins expressed by primary AML blasts. Primary patient samples were stimulated <i>in vitro</i> with recombinant EGFL7 (rEGFL7) or anti-EGFL7 blocking antibody to assess alterations in downstream signaling and the ability to effect blast differentiation and survival. We treated three independent AML models with anti-EGFL7 or IgG1 control to determine whether anti-EGFL7 could prolong survival <i>in vivo</i>.</p>Results:<p>We found EGFL7 significantly binds several signaling proteins important for normal and malignant hematopoiesis including NOTCH. Stimulation of AML blasts with rEGFL7 reduced NOTCH intracellular domain and NOTCH target gene expression while treatment with an anti-EGFL7 blocking antibody resulted in reactivation of NOTCH signaling, increased differentiation, and apoptosis. Competitive ligand-binding assays showed rEGFL7 inhibits DELTA-like (DLL) 4-mediated NOTCH activation while anti-EGFL7 combined with DLL4 significantly increased NOTCH activation and induced apoptosis. Using three different AML mouse models, we demonstrated that <i>in vivo</i> treatment with anti-EGFL7 alone results in increased survival.</p>Conclusions:<p>Our data demonstrate that EGFL7 contributes to NOTCH silencing in AML by antagonizing canonical NOTCH ligand binding. Reactivation of NOTCH signaling <i>in vivo</i> using anti-EGFL7 results in prolonged survival of leukemic mice, supporting the use of EGFL7 as a novel therapeutic target in AML.</p></div>

<p>Additional Supplementary Data</p>

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.