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

Nungruthai Nilsri,Ponlapat Rojnuckarin,Nipan Israsena,Panchalee Jangprasert,Jaturawat Pawinwongchai

doi:10.1038/s41598-021-83895-6

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.

Highlights

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders caused by acquired activating mutations of cytokine signal transduction resulting in excessive cellular proliferation
The modified induced pluripotent stem cells (iPSCs) were tested for Janus kinase 2 (JAK2) gene insertion by conventional polymerase chain reaction (PCR) using specific primers to JAK2-mutated vectors
Modified iPSCs demonstrated the PCR products representing an inserted JAK2 gene, whereas they were absent in normal iPSCs (Fig. 1A)

Summary

Introduction

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders caused by acquired activating mutations of cytokine signal transduction resulting in excessive cellular proliferation. The BCR-ABL fusion gene is the hallmark of chronic myeloid leukemia, while the main pathogenesis of BCR-ABL-negative MPNs is overactivation of the JAK/STAT p athway[1,2]. Clinical syndromes of the latter group comprise polycythemia vera (PV) that is characterized by erythrocytosis, essential thrombocythemia (ET) that shows isolated thrombocytosis and primary myelofibrosis (PMF) that is typified by bone marrow fibrosis and splenomegaly from extramedullary hematopoiesis. IPSCs have potentials to be blood disease models which are probably closer to human physiology than cancer cell lines or animals. They may become cell sources for transfusion or immunotherapies in the future

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Results

Conclusion