Engineered Expression of JAK2V617F Drastically Changes the Response of Progenitor Cells to Erythropoietin and Partially Mimics the Polycythemia Expression Signature.

Abstract Polycythemia vera (PV) is a myeloproliferative disorder, consisting of an abnormal increase of red blood cells. Over 90% of PV patients harbor the JAK2V617F mutation. To determine the distinct role that JAK2V617F plays in the pathogenesis of PV, we transduced human bone marrow CD34+ cells with either wild-type (WT) or V617F (MT) JAK2 constructs, incubated them in cytokine mixtures designed to promote full myeloid outgrowth (+EPO) or the same mixture lacking erythropoietin (EPO) (−EPO). After 5 days of growth, flow cytometry showed MT cells had a 3-fold increase in erythroid differentiation in the presence of EPO compared to WT cells and demonstrated EPO-independent erythroid development. To ascertain whether MT JAK2 yielded an abnormal EPO response, Affymetrix array analysis was performed on WT and MT CD34+ cells +/−EPO. When we compared WT-EPO cells to MT-EPO cells, 83 genes were significantly different in expression by 1.5x or more. Next we compared WT+EPO to WT-EPO (710 genes), MT+EPO to MT-EPO (1247 genes) and contrasted these groups. 332 genes found in the JAK2 WT response were missing in EPO-treated JAK2V617F cells; 869 genes regulated in JAK2V617F were absent in the WT dataset. A set of 378 genes, part of the normal EPO response, were retained in V617F signaling, but 51 of these were expressed at significantly different levels in the WT and MT conditions and were therefore added to the abnormal response to EPO, now 920 genes. To determine which experimental conditions best modeled PV we tested which set had the best ability to identify PV patients among a group of 9 PV and 8 control samples which we had subjected to Affymetrix expression profiling. The predictor genes were selected from (a) the 83 genes in regulated by MT JAK2 in the absence of EPO, (b) the 920 genes that were aberrantly regulted MT cells were treated with EPO, (c) and the 332 representing the normal EPO response missing in V617F EPO response. The best predictors were 12 genes identified from the 920 gene set that correctly predicted all 17 samples and performed perfectly on cross-validation at a p-value of 0.05. Genes selected from the other groups performed poorly. These analyses indicate that the abnormal EPO response in JAK2V617F cells is more indicative of PV than the normal EPO response and that JAK2 mutation is a gain of function mutation at the genetic level, with a transcriptional readout exceeding that of normal EPO signaling. We next compared the V617F EPO response to the expression profile of PV CD34+ cells and identified 210 in common out of 985 on the PV list (16%) showing a modest overlap. The largest ontological category in this set consisted of 40 genes and represents genes involved in cell growth and maintenance. Two additional well-represented categories, nucleic acid metabolism (26 genes) and protein metabolism (22 genes), are also involved in basic cellular functioning, indicating a profound state of dysfunction in PV cells harboring the V617F mutation. While myelopoiesis was similar between WT and MT cells, gene ontology analysis showed a deficit in immune response genes suggesting JAK2V617F may cause qualitative defects in myelopoiesis. Examination of these genes will illuminate alternative pathways, aberrantly regulated by V617FJAK2, involved in PV pathogenesis.