Epidermal Growth Factor-like 7 As a Novel Therapeutic Target in Mantle Cell Lymphoma
Authors: Goda Chinmayee , Kolovich Sofia , Kulkarni Rohan SUDHIR , Karunasiri Malith , Ruddich Alex , Nauman Eric , Singh Satishkumar , Rajgolikar Girish , Garzon Ramiro , Sehgal Lalit , Dorrance Adrienne M. 
Published: November 2021

Abstract

Mantle cell lymphoma (MCL) is an aggressive form of mature B-cell non-Hodgkin's lymphoma (NHL), accounting for nearly 6% of NHL cases. Currently, MCL patients are treated with aggressive chemo-immunotherapy regimens followed mostly by consolidation with autologous stem cell transplantation and maintenance rituximab. Despite these intensive therapies, MCL prognosis remains poor, with a median overall survival of 6-7 years, with most of the patients developing the refractory or recurrent disease. Thus, there is a need for novel and more effective, less toxic therapies for MCL. Epidermal growth factor-like 7 (EGFL7) is a protein secreted by endothelial cells and plays a critical role in angiogenesis. Our lab was the first to demonstrate a role for EGFL7 in hematologic malignancies, demonstrating that EGFL7 is increased in leukemic blasts of AML patients and that anti-EGFL7 treatment alone results in prolonged survival of leukemic mice (Papaioannou et al., 2017). While EGFL7 has been shown to play a role in some hematological malignancies, its role in MCL has not been investigated. Therefore, we assessed EGFL7 expression levels in MCL patients compared to healthy controls using the publicly available dataset GSE46846. We found significant increases in EGFL7 in malignant B cells from MCL patients compared to healthy individuals (p&lt;0.05). Furthermore, using the publicly available clinical data set (Scott et al., 2017), we found that MCL patients (n=122) with high EGFL7 expression associated with lower overall survival rates compared to MCL patients with low EGFL7 (24 months; vs. 48 months, respectively, p= 0.0057) (Figure 1).
To examine the therapeutic potential of targeting EGFL7 in MCL cells, we treated patient-derived xenograft (PDX) cells (n=3) with an anti-EGFL7 blocking antibody (Parsatuzumab) in vitro. We found an increase in apoptosis of MCL PDX cells compared to IgG control (15-50% vs. 0.5-2.4%, respectively), p&lt;0.0001. Similar results were found when treating three MCL cell lines (Rec1, Jeko1, and SP53) with anti-EGFL7 or control. We found a decrease in cell proliferation (20 vs. 70%, p&lt;0.0001) and an increase in apoptosis (67-87% vs. 8-17%, p&lt;0.0001) at 48-hours post-anti-EGFL7 treatment compared to IgG, respectively. Next, to determine whether anti-EGFL7 treatment could target MCL cells in vivo, NSG mice were subcutaneously injected with Rec1 cells (5 x10 6). Seven days post-injection, mice were treated with anti-EGFL7 or IgG (50mg/kg, three times/week) (n=5 per group). Tumors were measured every week, and mice were sacrificed when they reached end point criteria. We found that anti-EGFL7 treated mice had significantly decreased tumor volume than IgG (1116.58mm 3 vs. 3626mm 3, respectively, p=0.0116) and increased survival (p = 0.0034). Overall, our data show that targeting EGFL7 using an anti-EGFL7 blocking antibody inhibits MCL cell growth and prolongs survival in mouse models of MCL.
Our lab has previously shown that EGFL7 binds to the Epidermal growth factor receptor (EGFR) in AML (Bill et al., 2020). Knowing the importance of EGFR in lymphoma, we validated the binding of EGFL7 to EGFR in MCL cells by performing an immunoprecipitation (IP) assay on protein lysates from PDX cells (n=2) and Jeko1 cells. We found that EGFL7 protein was significantly enriched in protein fractions pulled down using anti-EGFR antibody compared to IgG. Conversely, we transfected Jeko1 cells with Flag-tagged EGFL7 plasmid and performed IP using anti-Flag antibody. EGFR protein was significantly enriched in the protein fraction pulled down using an anti-Flag antibody compared to IgG. Next, we examined the association between EGFL7 and EGFR expression in primary MCL patients and found that EGFR positively correlates with EGFL7 expression (n=122, r=0.1533). Further, Anti-EGFL7 treatment decreased phospho-AKT protein levels in PDX cells and MCL cell lines compared to IgG control, suggesting blocking EGFL7 abrogates EGFR mediated downstream signals.
In conclusion, this is the first report describing a role for EGFL7 in MCL growth and/or survival by modulating the EGFR-AKT signaling pathway and targeting EGFL7 using an anti-EGFL7 blocking antibody as a novel treatment to improve the outcome for MCL patients.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.