Transcriptomic and phospho-proteomic analyzes of erythroblasts expanded in vitro from normal donors and from patients with polycythemia vera

Abstract

Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days. Normal ERYs required exposure to the glucocorticoid dexamethasone (Dex) for expansion, while PV-derived ERYs expanded in the absence of dexamethasone. RNA expression profiling revealed enrichment of two known oncogenes, GPR56 and RAB4a, in PV-derived ERYs along with reduced expression levels of transcription factor TAL1 (ANOVA FDR < 0.05). While both normal and polycythemic-derived ERYs integrated signaling cascades for growth, they did so via different signaling pathways which are represented by their differential phospho-profiles. Our results show that normal ERYs displayed greater levels of phosphorylation of EGFR, PDGFRβ, TGFβ, and cKit, while PV-derived ERYs were characterized by increased phosphorylation of cytoplasmic kinases in the JAK/STAT, PI3K, and GATA1 pathways. Together these data suggest that PV erythroblast expansion and maturation may be maintained and enriched in the absence of dexamethasone through reduced TAL1 expression and by accessing additional signaling cascades. Members of this acquired repertoire may provide important insight into the pathogenesis of aberrant erythropoiesis in myeloproliferative neoplasms such as polycythemia vera.

Figure 1. Characterization of erythroid cells generated by day 10–13 in cultures of normal donors (ND) or polycythemia vera (PV) patients

Log₁₀ scatter plots of CD235a with either CD36 or CD45 of cells obtained at day 10, 11 and 13 after culture of mono-nuclear cells from normal healthy donors or PV patients stimulated with 100 ng/mL SCF, 3 Units/mL EPO and 1 ng/mL IL-3 in the absence of Dexamethasone. May-Grunwald staining of representative cytospun day 10 cells is also presented. These data show the greater percentage of ERY expressing CD36 but not CD235a and the marginally detectable levels of polychromatophilic/orthochromatic ERY in cultures of ND with Dex than in those without Dex. By contrast the frequency of ERY expressing CD36/CD235a was high and of polychromatophilic/orthochromatic ERY were barely detectable in cultures of PV cells without Dex. Black, blue, green and red arrows/arrowheads indicate pro-erythroblasts and basophilic, polychromatophilic and orthocromatic erythroblasts respectively. n.d. = not done.

Figure 2. Microarray analysis of erythroblasts expanded from mono-nuclear cells from normal donors and patients having polycythemia vera

Normal healthy derived erythroblasts expanded from mono-nuclear cells stimulated for 11–13 days with 10 ng/mL SCF, 3 Units/mL EPO and 1 ng/mL IL-3 and 10 uM Dexamethasone (Dex) and Polycythemia vera derived erythroblasts expanded from mono-nuclear cells stimulated with 10 ng/mL SCF, 3 Units/mL EPO and 1 ng/mL IL-3 without dexamethasone. Hypothesis testing using ANOVA with a false-discovery threshold of 0.05 reveals 55 probe sets which are differentially expressed between erythroblasts derived from normal healthy donors and polycythemic patients as shown by hierarchical clustering.

Figure 3. Reverse Phase Protein Array of erythroblast protein lysates collected from normal donors and patients with Jak2 V617F positive polycythemia vera

0.125–0.5 µg/µl of lysates were printed onto nitrocellulose-coated glass slides in triplicate and probed with a library of approximately 180 antibodies recognizing total, cleaved and phospho-protein endpoints. Primary antibody binding was detected using a biotinylated goat anti-rabbit IgG H+L (1:7500) or rabbit anti-mouse IgG (1:10) followed by streptavidin-conjugated IRDye680 fluorophore reaction. Differentially enriched signal intensities (Wilcoxon Rank-Test p < 0.05) of 39 features involved in multiple proliferation and survival pathways are shown.