title: 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.
authors.affiliation: Human Oncology and Pathogenesis Program and Leukemia Service, Memorial Sloan-Kettering Cancer Center, New York, NY 10029, USA.
authors.forename: Todd
authors.initials: T
authors.lastname: Hricik
authors.forename: Giulia
authors.initials: G
authors.lastname: Federici
authors.forename: Ann
authors.initials: A
authors.lastname: Zeuner
authors.forename: Giuliana
authors.initials: G
authors.lastname: Alimena
authors.forename: Agostino
authors.initials: A
authors.lastname: Tafuri
authors.forename: Valentina
authors.initials: V
authors.lastname: Tirelli
authors.forename: Lilian
authors.initials: L
authors.lastname: Varricchio
authors.forename: Francesca
authors.initials: F
authors.lastname: Masiello
authors.forename: Fiorella
authors.initials: F
authors.lastname: Ciaffoni
authors.forename: Stefania
authors.initials: S
authors.lastname: Vaglio
authors.forename: Emanuel F
authors.initials: EF
authors.lastname: Petricoin
authors.forename: Gabriella
authors.initials: G
authors.lastname: Girelli
authors.forename: Ross L
authors.initials: RL
authors.lastname: Levine
authors.forename: Anna Rita F
authors.initials: AR
authors.lastname: Migliaccio
mesh_terms: D000328:Adult; D000368:Aged; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D016022:Case-Control Studies; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D004900:Erythroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D004920:Erythropoiesis / Q000235:genetics*; D004921:Erythropoietin / Q000494:pharmacology; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D011087:Polycythemia Vera / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D040901:Proteomics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D019089:Stem Cell Factor / Q000494:pharmacology; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D020695:rab4 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D003907:Dexamethasone; D004921:Erythropoietin; D007377:Interleukin-3; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D043562:Receptors, G-Protein-Coupled; D019089:Stem Cell Factor; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D020695:rab4 GTP-Binding Proteins; C118925:ADGRG1 protein, human; C554270:EPO protein, human; C508596:IL3 protein, human; C067143:TAL1 protein, human