Page 36 - Haematologica-April 2018

F. Pasquier et al.
and STAT5 activation also persisted for 4 h after removal
of erythropoietin from the PFCP cells, whereas it was much more transient in control cells (Figure 1F).
Here we show that EPOR c.1300dup (p.Gln434Profs*11) is a strong gain-of-function mutation, which induces major erythropoietin hypersensitivity in primary ery- throid progenitors, similar to that observed in patients with polycythemia vera, as well as constitutive and per- sistent activation of JAK2 and STAT5.
Functional analysis of EPOR c.1300dup (p.Gln434Profs*11) in the Ba/F3 cell line
In order to study the functional impact of the new frameshift mutant, Ba/F3 cells were transduced to express different HA-tagged human EPOR: the wild-type receptor (EPOR WT), EPOR p.Gln434Profs*11 (EPOR FS), identical to the patient’s mutation or EPOR p.Gln444* (EPOR STOP) generating a truncated receptor at position 444 (Table 1, Figure 2A). EPOR FS and EPOR STOP differed by the nature of the C-terminal ten amino acid residues, namely the new sequence PALASMDTVP in EPOR FS and the natural sequence QLLRPWTLCP in EPOR STOP. These cells expressed quite similar levels of exogenous EPOR, as detected with anti-HA antibody (Figure 2C). Interestingly, EPOR FS migrated slightly above EPOR STOP, suggesting differences in post-trans- lational modification. However, the glycosylation states of EPOR WT, EPOR STOP and EPOR FS were similar after using Endo H and PGNase F (Online Supplementary Figure S1).
MTT-like assays were performed to investigate the potential effects of EPOR STOP and EPOR FS on cell prolif- eration. None of these mutants was able to induce cytokine-independent cell growth. However, EPOR FS con- ferred a 4- to 5-fold greater erythropoietin hypersensitivity to Ba/F3 cells compared to EPOR WT. Interestingly the ery- thropoietin-induced growth of EPOR STOP Ba/F3 cells was similar to that of EPOR WT cells (Figure 2B). To confirm these results in a human setting, we transduced EPOR WT, STOP and FS in the human UT-7 cell line, which expresses endogenous EPOR and found similar results as in the Ba/F3 cell line (Online Supplementary Figure S2A).
We checked the signaling pathways and observed that EPOR FS induced constitutive phosphorylation of STAT5, AKT and ERK compared to EPOR WT and to a higher extent than EPOR STOP in Ba/F3 cells (Figure 2C) and UT- 7 cells (Online Supplementary Figure S2B). Semi-quantitative analysis of spontaneous STAT5 phosphorylation showed a significant increase in EPOR FS compared to EPOR WT and EPOR STOP (Figure 2D). Moreover, we observed a similar persistent STAT5 activation in both EPOR FS and EPOR STOP Ba/F3 cells compared to EPOR WT cells (4 h, 2 h and 30 min, respectively, after erythropoietin removal) (Figure 2E).
These results show that EPOR FS confers a similar ery- thropoietin hypersensitivity to Ba/F3 and UT-7 cells as that observed in PFCP erythroid progenitors, while the truncated mutant EPOR STOP did not confer such ery- thropoietin hypersensitivity to these cells. The greater erythropoietin hypersensitivity induced by EPOR p.Gln434Profs*11 cannot, therefore, be explained by the receptor truncation itself and the loss of the two SHP-1 and SOCS3 binding sites which are responsible for a per- sistent activation, but rather by the appearance of a new C-terminal tail that confers spontaneous signaling.
Effects of the c.1300dup (p.Gln434Profs*11) mutation on erythropoietin receptor stability, cell sur- face expression and dimerization
To further characterize this new EPOR mutant, the cell surface expression of the wild-type receptor and both mutants was studied by flow cytometry. At similar levels of GFP (transduction of cells with IRES-GFP retroviruses), EPOR FS was significantly more abundant at the cell sur- face (more than 2-fold, P=0.0002) than EPOR WT and EPOR STOP in both Ba/F3 and UT-7 cell lines (Figure 3A and Online Supplementary Figure S3). These results were further confirmed in Ba/F3 cells using radiolabeled 125I-ery- thropoietin (2,412±494 receptors for EPOR FS, P=0.015, 1,018±284 receptors for EPOR STOP and 1,201±304 receptors for EPOR WT) (Figure 3b). We next investigated the stability of the receptors using treatment with the pro- tein synthesis inhibitor cycloheximide. EPOR FS was more stable than both EPOR WT and EPOR STOP (half- life of 2 h and 1 h, respectively) (Figure 3C). We also stud- ied the dimerization of human EPOR monomers by split Gaussia luciferase assay in steady-state conditions in HEK cells, in the absence of erythropoietin (Figure 3D). Close proximity between the C-terminal cytosolic domains of EPOR was increased by 4-fold with EPOR FS compared to control and EPOR STOP, as assessed by reconstitution of the split Gaussia luciferase activity (Figure 3E).
The increased stability and cell surface localization of EPOR FS may also result in a defect in the receptor inter- nalization pattern due to the loss of specific domains located in the C-terminal part of the wild-type receptor. However, EPOR WT, STOP and FS displayed the same internalization pattern as assessed by 125I-erythropoietin labeling experiments (Figure 4A,B). Furthermore, we noted that a dileucine motif, known to be a potential clathrin-dependent endocytosis signal,37 was lost in the new C-terminal tail of the EPOR FS. We assumed that this particular modification could be involved in the increased cell surface expression of EPOR FS. Thus, based on EPOR WT and EPOR STOP constructs, we generated two other mutants, EPOR WT/diL and EPOR STOP/diL, where the dileucine motif was removed (Figure 4C). However its abrogation did not modify either the erythropoietin sensi- tivity of EPOR WT or STOP Ba/F3 cells (Figure 4D) or the localization of the receptors at the cell surface (Figure 4E). Likewise, EPOR WT/diL and EPOR STOP/diL Ba/F3 cells displayed a similar signaling pattern to that of EPOR WT and EPOR STOP cells, respectively (Figure 4F).
Collectively these results show that p.Gln434Profs*11 increases EPOR stability, dimerization and localization at the cell surface without modifying internalization of the receptor.
Different mechanisms are involved in the erythropoietin hypersensitivity phenotype in primary erythrocytosis according to the type of EPOR mutation
We wondered whether this model could be extended to other EPOR mutations already described in PFCP. We therefore investigated the impact of the frameshift EPOR c.1311_1312del (p.Pro438Metfs*6) mutation25 and its designed nonsense mutant counterpart, EPOR c.1327_1329delinsTAA (p.Pro443*) (Figure 5A) on the pro- liferation rate of Ba/F3 cells. These mutants lack the last 65 amino acids of the receptor, retaining two of the eight conserved tyrosine residues, Tyr-368 and Tyr-426 (Table 1, Figure 6A,C,D). Interestingly, EPOR p.Pro438Metfs*6 and
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