Erythroferrone (ERFE) Is Selectively Expressed in Human CD71+ Erythroprogenitor Cells and Deregulated Overexpression Is Associated with a Favorable Outcome in Low Risk Myelodysplastic Syndrome (MDS)

Introduction

Recently, Erythroferrone (ERFE) was discovered as a new regulator of hepcidin in the context of hematopoietic stress and erythropoietin (EPO) stimulation (Kautz et al., Nature Genetics 2014). ERFE has been shown to be expressed by erythroprogenitor cells of the bone marrow in response to increased erythroid activity induced by phlebotomy, EPO treatment or simulation of infectious situations in mice. It induces increased iron availability by downregulation of hepcidin in the liver and therefore represents an important new factor in iron homeostasis to be explored as a potential diagnostic or therapeutic target in the context of anemia and iron overload. Myelodysplastic Syndromes (MDS) are a group of heterogeneous malignant hematologic diseases characterized by inefficient hematopoiesis, severe anemia and deregulated iron homeostasis. In order to determine the specific role of ERFE in MDS, we analyzed the gene expression of ERFE in different hematopoietic compartments of MDS patients and healthy controls and correlated the differential expression data with clinical parameters and survival.

Methods

CD71+ erythroprogenitor cells (n=198 samples) were immunomagnetically purified from mononuclear bone marrow (BM) cells of a total of n=148 MDS and n=18 sAML patients. Chronological samples were available in n=21 cases. For controls, CD71+ BM cells were analyzed from n=35 healthy donors. In addition to CD71+ cells, CD61+, CD15+ , CD34+, selected from 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 volunteers. After total RNA extraction 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.

Results

In comparative expression analyses of different hematopoietic BM progenitor fractions (CD34+, CD15+, CD61+ and CD71+), ERFE was almost exclusively expressed in the erythropoietic CD71+ compartment. ERFE expression profiles in the CD71+ subset revealed a highly significant overexpression of this gene in MDS IPSS-low/int-1-risk (fold change (FC)=4.3, p<0.0001), IPSS-int-2/high-risk (FC=6.23, p<0.0001) and sAML (FC=6.69, p<0.0001) relative to healthy controls. ERFE expression profiles in MDS and sAML did not correlate with clinical laboratory parameters such as hemoglobin, EPO levels, ferritin, cobalamine, folic acid, transferrin, transferrin saturation, soluble transferrin receptor, reticulocytes, zinc protoporphyrin and lactate dehydrogenase. A negative correlation was observable for c-reactive protein levels (p=0.0053, Spearman r=-0.29) suggesting a possible link between an inflammatory environment and ERFE regulation. In exemplary chronological time course samples, ERFE expression was upregulated subsequent to clinical therapies such as 5-Azacytidine or Lenalidomide. Interestingly, in the total cohort of MDS patients with survival data follow up (n=90), low ERFE expression was associated with a significantly worse survival than high ERFE expression (median survival 2.1 years versus not reached, HR: 4.4, p=0.0007). This observation was even more pronounced in the subgroup analysis of MDS IPSS low/int-1 risk patients (n=54, median survival 2.1 years versus not reached, HR: 22, p<0.0001).

Conclusion

The observed highly aberrant overexpression of ERFE in CD71+ erythropoietic progenitor cells suggests an important role for this gene in the dysfunctional erythropoiesis of MDS. The observation of a correlation between ERFE expression and survival, especially in low risk MDS patients with no apparent coherence to other established clinical markers warrants further pursuit of ERFE expression profiles in CD71+ BM cells of MDS patients as a possible independent prognostic marker. Moreover, aberrant levels of ERFE could provide a promising target for novel therapeutic avenues that mechanistically address dysfunctional erythropoiesis in MDS.