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F. Pasquier et al.
clonal and PFCP patients do not show any risk of their dis-
ease transforming into myelofibrosis or leukemia. Moreover, somatic JAK2V617F15 and JAK2 exon1216 muta- tions, hallmarks of polycythemia vera, are not found in PFCP,12,13 which is, however, characterized by the presence of germline mutations in the erythropoietin receptor gene (EPOR).
Erythropoietin receptor (EPOR) is a type I cytokine receptor,17 essentially expressed by erythroid progenitors in hematopoietic tissue. The binding of erythropoietin to its receptor at the cell surface leads to the transient activa- tion of preformed EPOR-JAK2 complexes18,19 and down- stream signaling pathways, including signal transducer and activators of transcription (STAT),20 phosphatidylinos- itol 3 kinase/Akt21 and mitogen-activated protein kinase pathways. Erythropoietin-induced signaling is crucial for the proliferation and the survival of erythroid progenitors as well as for terminal erythroid differentiation.22
Around 20 germline heterozygous nonsense and frameshift mutations located in exon 8 of EPOR have been described so far in PFCP, all leading to the truncation of the C-terminal part of the receptor.3-11,13,14,23-28 Interestingly, sim- ilar EPOR truncations have been described in BCR-ABL1- like acute lymphoblastic leukemia, due to acquired rearrangements of EPOR with immunoglobulin chain loci.29 The C-terminal part of the receptor includes several conserved tyrosine residues that are docking sites for pos- itive and negative regulators of EPOR signaling. The ery- thropoietin hypersensitivity of PFCP progenitors is, there- fore, usually explained by the disappearance of negative regulatory domains located in the truncated part of the receptor.30,31 A few missense EPOR mutations have also been described, but their involvement in the PFCP pheno- type is not yet clear.4,7,13,26-28,32 Previous studies have suggest- ed that truncated EPOR mutations might not be equiva- lent in term of underlying mechanisms leading to EPOR activation,14,27 but relatively few functional studies have been carried out. We, therefore, investigated the mecha- nism of some EPOR mutations in PFCP.
We identified and extensively studied a new germline frameshift EPOR mutation, c.1300dup (p.Gln434Profs*11), responsible for marked erythropoietin hypersensitivity as in JAK2V617F-positive polycythemia vera. We modeled EPOR p.Gln434Profs*11 and several other EPOR mutants already described in the Ba/F3 and UT-7 cell line and demonstrated that different mechanisms are involved in the erythropoietin hypersensitivity phenotype, according to the type of EPOR mutation, highlighting that PFCP is a more complex pathology than usually considered.
Methods
Materials
Human recombinant erythropoietin and interleukin-3 were generous gifts from Amgen (Neuilly, France). Stem cell factor was purchased from Biovitrum AB (Stockholm, Sweden).
Patients, cell purification and erythroblast cultures
Peripheral blood samples from the patient or healthy donors were collected by leukapheresis. Written informed consent was obtained from the patient in accordance with the Declaration of Helsinki and the study was approved by the ethics committee of La Pitié-Salpétrière Hospital. Mononuclear cells were separated over a Ficoll density gradient and CD34+ cells were purified by a
double-positive magnetic cell sorting system (AutoMACS; Miltenyi Biotec, Paris, France). CD34+ cells were amplified in ery- throid conditions for 7-10 days in Iscove modified Dulbecco medi- um with penicillin/streptomycin/glutamine, alpha-thioglycerol, bovine serum albumin, a mixture of sonicated lipids and insulin- transferrin in the presence of recombinant human cytokines (25 ng/mL stem cell factor, 100 U/mL interleukin-3, 1 U/mL erythro- poietin).
EPOR sequencing
Genomic DNA was extracted from blood, nails and hair using
standard procedures. Exon 8 of EPOR was amplified and sequenced in both directions. Mutations are numbered as recom- mended by the Human Genome Variation Society (http://www.hgvs.org/) using the reference sequence NM_000121.3. Participants gave written informed consent to the genetic study.
Quantification of clonogenic progenitors in semi-solid cultures
Colony assays were performed with 500 purified CD34+ pro- genitors per culture dish in duplicate in H4100 Methocult media (StemCell Technologies, Grenoble, France) supplemented with 12% bovine serum albumin, 30% or no fetal bovine serum, 2-β- mercaptoethanol (1 mM), 1% L-glutamine, stem cell factor (25 ng/mL), interleukin-3 (100 U/mL) and in the absence or presence of increasing concentrations of erythropoietin (0.001; 0.01; 0.1 and 1 U/mL). Burst-forming units-erythroid (BFU-E)-derived colonies were counted on day 14.
DNA manipulation and retrovirus production
EPOR mutations were introduced into the pMX-HA-human EPOR WT-IRES-GFP plasmid by the QuikChange site-directed mutagenesis method using the PfuUltra high-fidelity DNA poly- merase (Agilent Technologies, Stratagene, Les Ulis, France): c.1300dup (p.Gln434Profs*11, EPOR FS); c.1330G>T (p.Gln444*, EPOR STOP); c.1303_1304delinsGC (p.Leu435Ala, EPOR WTdiL or EPOR STOPdiL); c.1195G>T (p.Glu399*); c.1273G>T (p.Glu425*); c.1311_1312del (p.Pro438Metfs*6); c.1327_1329delinsTAA (p.Pro443*); c.[1300dup; 1311G>C] (p.[Gln434Profs*11; Ala437Arg]) (Table 1). Full-length EPOR mutant cDNA was verified by sequencing.
Cell lines
The murine Ba/F3 and human UT-7 cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum (StemCell Technologies, Grenoble, France) and 5% WEHI-conditioned medium as a source of murine interleukin-3 or granulocyte-macrophage colony-stimulating factor (10 ng/mL). Cells were transduced with pMX-IRES-GFP retrovirus to stably express the human wild-type EPOR or EPOR mutants, carrying a N-terminal HA-tag. GFP+ cells were subsequently sorted by flow cytometry (Influx, Beckton-Dickinson, Le Pont-de-claix, France).
Proliferation assays
The premixed WST-1 cell proliferation assay was carried out according to the manufacturer’s instructions (Takara Bio Europe, Clontech, Saint-Germain-en-Laye, France). Experiments were per- formed in triplicate. Dose-response curves to erythropoietin were expressed as percentages of viability of the maximal response.
Western blot analysis
For signaling studies, cells were starved for 5 h and then incu- bated with increasing concentrations of erythropoietin for 15 min or stimulated with 1 U/mL erythropoietin for 15 min and washed
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