EGFL7 Antagonizes NOTCH Signaling and Represents a Novel Therapeutic Target in Acute Myeloid Leukemia.

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.

Clinical and functional significance of circular RNAs in cytogenetically normal AML.

Circular RNAs (circRNAs) are noncoding RNA molecules that display a perturbed arrangement of exons, called backsplicing. To examine the prognostic and biologic significance of circRNA expression in cytogenetically normal acute myeloid leukemia (CN-AML), we conducted whole-transcriptome profiling in 365 younger adults (age 18-60 years) with CN-AML. We applied a novel pipeline, called Massive Scan for circRNA, to identify and quantify circRNA expression. We validated the high sensitivity and specificity of our pipeline by performing RNase R treatment and RNA sequencing in samples of AML patients and cell lines. Unsupervised clustering analyses identified 3 distinct circRNA expression-based clusters with different frequencies of clinical and molecular features. After dividing our cohort into training and validation data sets, we identified 4 circRNAs (circCFLAR, circKLHL8, circSMC1A, and circFCHO2) that were prognostic in both data sets; high expression of each prognostic circRNA was associated with longer disease-free, overall, and event-free survival. In multivariable analyses, high circKLHL8 and high circFCHO2 expression were independently associated with better clinical outcome of CN-AML patients, after adjusting for other covariates. To examine the biologic relevance of circRNA expression, we performed knockdown screening experiments in a subset of prognostic and gene mutation-related candidate circRNAs. We identified circFBXW7, but not its linear messenger RNA, as a regulator of the proliferative capacity of AML blasts. In summary, our findings underscore the molecular associations, prognostic significance, and functional relevance of circRNA expression in CN-AML.

Author Correction: The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Expression and functional relevance of long non-coding RNAs in acute myeloid leukemia stem cells.

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.

The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

Long non-coding RNAs (lncRNAs) are important regulatory molecules that are implicated in cellular physiology and pathology. In this work, we dissect the functional role of the HOXB-AS3 lncRNA in patients with NPM1-mutated (NPM1mut) acute myeloid leukemia (AML). We show that HOXB-AS3 regulates the proliferative capacity of NPM1mut AML blasts in vitro and in vivo. HOXB-AS3 is shown to interact with the ErbB3-binding protein 1 (EBP1) and guide EBP1 to the ribosomal DNA locus. Via this mechanism, HOXB-AS3 regulates ribosomal RNA transcription and de novo protein synthesis. We propose that in the context of NPM1 mutations, HOXB-AS3 overexpression acts as a compensatory mechanism, which allows adequate protein production in leukemic blasts.