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

Abstract

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.

Recent Progress in Interferon Therapy for Myeloid Malignancies

“…Current understanding suggests IFN-a-mediated promotion of JAK2 V617F-mutated HSC cell cycle not only exhausts the cells, but uses induces differentiation to the erythroid lineage, making them susceptible to IFN-a-mediated apoptosis (120). Nevertheless, the effects of IFN-a demonstrated here may not be wholly specific to the V617F mutant, since stem cell derived erythrocytes containing JAK2 with exon 12 mutations (also causing constitutive JAK2 activation, as seen in PV) were also sensitive to IFN-a treatment (121).…”

Induced Pluripotent Stem Cells as a Tool for Modeling Hematologic Disorders and as a Potential Source for Cell-Based Therapies

“…JAK2V617F-iPSCs did not exhibit a significant increase in erythroid cell proliferation or differentiation, most likely because the studied JAK2V617F expression was more consistent with the heterozygous JAK2V617F mutation in ET patients, where the endogenous JAK2 gene was still present [148,149]. The utilization of different types of JAK2 mutations in patient-derived iPSCs or generated mutant iPSCs for the discovery of pathogenesis and candidate therapy for MPNs has been demonstrated [149,150]. Additionally, hiPSCs derived from patients with the calreticulin (CALR) gene mutation, which is usually found in ET and primary myelofibrosis patients, could reflect disease phenotypes by representing megakaryopoiesis and prominent colony-forming unit megakaryocytes (CFU-MK) [151,152].…”

“…JAK2exon12-iPSCs, but not JAK2V617F-iPSCs, exhibited an increased erythropoiesis resembling the pathophysiology in PV patients. JAK2V617F-iPSCs did not exhibit a significant increase in erythroid cell proliferation or differentiation, most likely because the studied JAK2V617F expression was more consistent with the heterozygous JAK2V617F mutation in ET patients, where the endogenous JAK2 gene was still present [148,149]. The utilization of different types of JAK2 mutations in patient-derived iPSCs or generated mutant iPSCs for the discovery of pathogenesis and candidate therapy for MPNs has been demonstrated [149,150].…”

Stem cell factor and erythropoietin-independent production of cultured reticulocytes

“…By contrast, mutations in Jak2 exon 12, which are much rarer than the V617F mutation, are much more specifically associated with PV. Experiments in which both mutations have been introduced in iPSCs have shown that, compared with JAK2V617F-iPSCs, JAK2exon12-iPSCs produced a greater number of erythroid cells that displayed more mature morphology and expressed more adult hemoglobin 50 . Importantly, exon12 mutations led to significantly higher levels of phospho-STAT1 but lower phospho-STAT3 s compared with JAK2V617F-iPSCs in response to erythropoietin.…”