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Abstract An understanding of cellular events that are propagated within erythroid progenitor cells upon HGF-R / JAK complex activation is of basic importance for generating new insight into regulated red cell formation, anemia and myeloproliferative disease. Using the EPO/EPOR/JAK2 system as a paradigm, our group is successfully applying post-translational modification-based proteomics to uncover important new mediators of EPO-dependent human erythropoiesis (certain of which may also relate to EPO's untoward effects on hypertension and cancer progression). Here, we report on the discovery of a novel ORF, "C1ORF150", that is strongly tyrosine phosphorylated in response to EPO, possesses several unique features, and modulates EPO- dependent erythroid progenitor cell formation. In human erythroid progenitor UT7epo cells, EPOR ligation leads to C1ORF150 phosphorylation at tandem tyrosine p-Y69, p-Y89 and p-Y110, p-Y129 sites (up to 10-fold within 15 minutes). For each PTM site, EPOR/JAK2 mediated- phosphorylation was validated in independent LC-MS/MS experiments using Hematide as an EPOR agonist. p-Y69 and p-Y89 are predicted SFK sites, while p-Y110 and p-Y129 are predicted RTK sites (including KIT). Notably, C1ORF150 is conserved in H. sapiens and primates, but is not represented in mouse, rat or lower vertebrates. In addition, C1ORF150 has no obvious orthologues, but within EPO-regulated pY regions exhibits sequence homology with HGAL, an important factor for B cell receptor signaling. To assess C1ORF150's functional effects, we used a lentiviral shRNA loss-of-function approach (80% knockdown efficiency). At physiological EPO levels, the knockdown of C1ORF150 substantially compromised UT7epo erythroid progenitor cell (EPC) survival, including 200% increases in apoptosis observed relative to control sh-NT transduced EPCs (p < 0.01). The ectopic expression of C1ORF150, in contrast, heightened baseline JAK2 activation, and potentiated STAT5 activation following EPO challenge. C1ORF150's subcellular localization proved to be predominantly membrane associated. With regards to expression profiles, C1ORF150 levels were markedly elevated in bone marrow (among 30 human tissues), and during erythroid development were maximal at a CFUe stage. Furthermore, transcriptome profiles of myelodysplastic syndrome (MDS) CD34pos hematopoietic progenitor cells revealed elevated C1ORF150 expression in MDS refractory anemia (p=0.005) and refractory anemia-blast patients (p=0.05) compared to normal controls. In summary, via PTM-proteomics we have discovered "C1ORF150" as a major new pY- regulated EPOR/JAK2 target and membrane associated phosphoprotein that is proposed to have evolved in human erythroid progenitor cells to support EPO's cytoprotective effects, and red cell formation, in part by reinforcing JAK2 and STAT5 activation. In anemia and pre-leukemic contexts, attention also is brought to possible roles for C1ORF150 in the onset and progression of MDS. Disclosures No relevant conflicts of interest to declare.