Mutated JAK2 signal transduction in human induced pluripotent stem cell (iPSC)-derived megakaryocytes.

Janus kinase 2 (JAK2) gene mutations are the main drivers for polycythemia vera (PV) and essential thrombocythemia (ET). The mechanisms of single altered gene causing two different diseases are unclear. Additionally, novel treatments specifically targeting mutated JAK2 proteins are needed. In this study, the induced pluripotent stem cells (iPSCs) were virally transduced to express wild-type JAK2 (JAK2WT), JAK2p.V617F (JAK2V617F) or JAK2p.N542_E543del (JAK2exon12) under a doxycycline-inducible system. The modified iPSCs which were differentiated into megakaryocytes in the presence vs. absence of doxycycline were compared to ensure that the differences were solely from mutated JAK2 expressions. The JAK2V617-expressing iPSCs yielded significantly higher numbers of megakaryocytes consistent with the ET phenotype, while there was no enhancement by JAK2exon12 expression compatible with the pure erythrocytosis in humans. Capillary Western analyses revealed significantly greater JAK2 phosphorylation in iPSCs carrying JAK2V617F but not in JAK2WT and JAK2exon12 iPSCs. Activation of STAT3, STAT5 and AKT was increased by JAK2V617F, while they were decreased in JAK2exon12 iPSCs. Notably, interferon alpha and/or arsenic trioxide inhibited megakaryocytes proliferation and reduced JAK2, STAT3, STAT5 and AKT phosphorylation in mutant JAK2-expressing iPSCs compared with those without induction. In conclusion, JAK2V617F expression in iPSCs preferentially promoted megakaryocytes with a signaling profile distinctive from JAK2exon12 expression. Treatments with interferon alpha or arsenic trioxide preferentially suppressed the mutated over wild-type JAK2 signaling. This iPSC model is helpful in mechanistic studies and novel therapy screen for myeloproliferative neoplasm.

Distinct effects of V617F and exon12-mutated JAK2 expressions on erythropoiesis in a human induced pluripotent stem cell (iPSC)-based model.

Activating mutations affecting the JAK-STAT signal transduction is the genetic driver of myeloproliferative neoplasms (MPNs) which comprise polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis. The JAK2p.V617F mutation can produce both erythrocytosis in PV and thrombocytosis in ET, while JAK2 exon 12 mutations cause only erythrocytosis. We hypothesized that these two mutations activated different intracellular signals. In this study, the induced pluripotent stem cells (iPSCs) were used to model JAK2-mutated MPNs. Normal iPSCs underwent lentiviral transduction to overexpress JAK2p.V617F or JAK2p.N542_E543del (JAK2exon12) under a doxycycline-inducible system. The modified iPSCs were differentiated into erythroid cells. Compared with JAK2V617F-iPSCs, JAK2exon12-iPSCs yielded more total CD71+GlycophorinA+ erythroid cells, displayed more mature morphology and expressed more adult hemoglobin after doxycycline induction. Capillary Western immunoassay revealed significantly higher phospho-STAT1 but lower phospho-STAT3 and lower Phospho-AKT in JAK2exon12-iPSCs compared with those of JAK2V617F-iPSCs in response to erythropoietin. Furthermore, interferon alpha and arsenic trioxide were tested on these modified iPSCs to explore their potentials for MPN therapy. Both agents preferentially inhibited proliferation and promoted apoptosis of the iPSCs expressing mutant JAK2 compared with those without doxycycline induction. In conclusion, the modified iPSC model can be used to investigate the mechanisms and search for new therapy of MPNs.