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.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Calcium-Binding Proteins/metabolism , EGF Family of Proteins/metabolism , Leukemia, Myeloid, Acute/pathology , Receptors, Notch/antagonists & inhibitors , Animals , Apoptosis , Calcium-Binding Proteins/genetics , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , EGF Family of Proteins/genetics , Female , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Receptors, Notch/metabolism , Signal TransductionABSTRACT
In acute myeloid leukemia (AML), novel therapies are needed to target not only the rapidly dividing AML blasts but also the distinct population of leukemia stem cells (LSCs), which have abnormal self-renewal capacity and increased chemotherapy resistance. Elucidation of the expression and function of deregulated genes in LSCs is critical to specifically target LSCs and may consequently lead to improving outcomes of AML patients. Here, we correlated long non-coding RNA (lncRNA) expression profiles obtained from two RNA-seq datasets of 375 younger (aged <60 years) 76 older (≥60 years) adults with cytogenetically normal AML with a 'core enriched' (CE) gene expression signature (GES) associated with LSCs. We identified a LSC-specific signature of 111 lncRNAs that correlated strongly with the CE-GES. Among the top upregulated LSC-associated lncRNAs, we identified the lncRNA DANCR. Further experiments confirmed that DANCR is upregulated in functionally validated LSC-enriched populations. DANCR knock-down in LSCs resulted in decreased stem-cell renewal and quiescence. Furthermore, we showed that targeting Dancr in vivo using a primary murine model of AML (expressing both Mll partial tandem duplication/Flt3 internal tandem duplication) prolonged the survival of mice after serial transplantation. Our data suggest that LSCs have a distinct lncRNA signature with functional relevance and therapeutic potential.
