Gene Expression of the Erythroid Regulator Erythroferrone (ERFE) is Highly Deregulated in CD71+ Erythroprogenitor Cells of Patients with Myelodysplastic Syndromes and Demonstrates Prognostic Relevance
Authors: Mossner Maximilian , Stöhr Alexandra , Nolte Florian , Jann Johann-Christoph , Fey Stephanie , Nowak Verena , Obländer Julia , Pressler Jovita , Baldus Claudia D , Schulze Torsten J , Neumann Martin , Hofmann Wolf-Karsten , Nowak Daniel 
Published: October 2019

Abstract
Introduction
Myelodysplastic Syndromes (MDS) are clonal hematologic diseases that are characterized by inefficient hematopoiesis, severe anemia and resulting deregulated iron homeostasis. Apart from supportive therapy with red blood cell transfusions some MDS patients with anemia respond to treatment with erythropoiesis stimulating agents such as Erythropoietin (EPO). However, the majority of these patients also become refractory to EPO treatment during the course of disease, suggesting a dysfunctional regulation of erythropoiesis downstream of EPO signaling in MDS. Most recently, a crucial erythroid regulator of iron metabolism named Erythroferrone (ERFE) was newly discovered, which is selectively produced by bone marrow (BM) erythroprogenitor cells during hematopoietic stress and EPO stimulation (Kautz et al. ASH plenary session 2013 and Kautz et al., Nature Genetics 2014). Aberrant expression of ERFE has been shown to directly result in critical impairment of erythropoiesis. We therefore sought to examine the role of ERFEexpression in CD71+ erythroprogenitor cells derived from patients with MDS and secondary acute myeloid leukemia (sAML).
Methods
CD71+ erythroprogenitor cells were immunomagnetically isolated from ficollized mononuclear BM cells of patients suffering from MDS (n=86, IPSS-low/int-1-risk n=69, IPSS-int-2/high-risk n=17), sAML (n=18) and age-matched healthy donors (n=17). In addition to CD71+ cells, CD34+, CD61+, CD15+ selected BM as well as CD3+ selected peripheral blood (PB) cells were immunomagnetically collected from three MDS patients as well as two healthy young and two healthy old donors. After total RNA was extracted using the AllPrep DNA/RNA Mini kit (Qiagen), cDNA was transcribed from RNA via Quantitect cDNA synthesis kit (Qiagen). Subsequently, ERFE expression was quantified from cDNA by quantitative PCR and normalized to corresponding GPIhousekeeping gene expression levels. Patient follow up (FU) data was available for n=55 MDS and n=14 sAML samples.
Results
Analysis of ERFE expression in CD34+, CD15+, CD61+ and CD71+ BM as well as CD3+ and unselected mononuclear PB cells from MDS patients and healthy donors revealed almost exclusive expression of ERFE in CD71+ erythroprogenitor cells irrespective of disease state. Our analysis of ERFE expression profiles in this specific cell subset revealed a highly significant overexpression of this gene in MDS IPSS-low/int-1-risk (fold change (FC)=4.1, p<0.0001), IPSS-int-2/high-risk (FC=4.6, p=0.0003) and sAML (FC=6.5, p<0.0001) relative to age-matched healthy controls. Despite this marked profile of aberrantly regulated ERFE we identified a distinct fraction of patients with expression levels similar or even lower than those measured in healthy donors in 20% (11/55) and 36% (5/14) of analyzed MDS and sAML cases with FU. Univariate analysis revealed that low abundance of CD71+ ERFE transcripts was significantly associated with inferior overall survival (OS) in MDS patients (median survival 1.7 years vs. not reached, p=0.0066) and also sAML (median survival 0.1 vs. 0.8 years, p=0.031).
Conclusion
The recent identification of the novel key regulatory gene ERFE in mouse models has greatly improved the understanding of the dynamic regulation of erythropoiesis. Our observation of almost exclusive ERFE expression in human BM erythroprogenitor cells further underlines its important role in human erythropoietic regulation both in healthy and myelodysplastic hematopoiesis. Moreover, strong upregulation of erythropoiesis stimulating ERFE in a large proportion of MDS patients usually suffering from anemia likely indicates its involvement in perturbed mechanisms of feedback signaling in MDS erythropoiesis. Pending integration with further clinical data, the current observation of significantly inferior survival probability for MDS and sAML patients with low ERFE expression levels indicates the potentially important biologic and clinical relevance of this novel regulatory gene in the pathogenesis of MDS. Consequently, aberrant levels of the erythroid hormone ERFEin MDS erythroprogenitor cells might provide a promising target for novel therapeutic avenues that mechanistically address dysfunctional erythropoiesis in MDS.

Disclosures
Nolte: Celgene Corp., Novartis Pharma: Honoraria, Research Funding.