Generation, Characterization and Genetic Modification of Human iPSCs Containing Calr, MPL and JAK2 Mutations Found in MPN Patients

Abstract Introduction: Myeloproliferative neoplasms (MPNs) that include polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are associated with multiple somatic and myeloid-specific mutations. A major challenge to the study of these diseases has been the lack of model systems that can accurately represent their complexity; the clonal heterogeneity within the hematopoietic stem/progenitor cells (HSPC) population is confounding and it is technically challenging to isolate and expand patient HSPC clones in vitro. To overcome this hurdle, we have utilized induced pluripotent stem cell (iPSC) technology to generate disease and clone-specific iPSC lines that preserve the genetic identity of HSPC clones with the JAK2-V617F mutation (Ye et al., 2009 in Blood). Recent advances in MPN genetics have identified CALR and MPL mutations in the majority of JAK2-V617F negative MPN. We therefore created additional panels of iPSCs from patients carrying CALR and MPL mutations in addition to our JAK2-mutated iPSCs. In addition, we used CRISPR/Cas9 technology to study genetic lesions and their contribution the phenotypes by using iPSC in vitro hematopoietic differentiation assays (Ye, et al., 2015 in Molecular Therapy). Methods: We generated iPSC lines from MPN patients with JAK2V617F, CALR or MPL mutations using peripheral blood CD34+ cells by transient expression of reprogramming factors in plasmid vectors. We characterized the pluripotency and karyotype of the derived iPSC lines, and determined if they contained the same somatic mutation as found in blood samples of each MPN patient. Then the derived iPSC cell lines were differentiated to CD34+ HSPCs, erythroid or megakaryocytic cell lineages using defined in vitro culture systems. Erythroid differentiation was determined by flow cytometric analysis of CD235a (Glycophrin A)-positive and CD45-negative cells. Megakaryocytic differentiation was determined by the levels of CD41+CD42+ cells. Results: In vitro differentiation assays have shown increased erythroid differentiation of HSPCs derived from patient-specific iPSCs that carry the JAK2V617F mutation, recapitulating a major PV disease feature. Editing a PV-derived JAK2V617F/V617F iPSC line to JAK2V617F/WT abrogated endogenous erythroid colony (EEC) formation, erythroid expansion and altered erythropoietin dose-response curve characteristic of the parent line. Conversely, gene editing a JAK2V617F/WT to a JAK2V617F/V617F iPSC line from a different PV patient conferred EEC formation and expanded erythropoiesis. In addition, data generated from these isogenic iPSCs suggested the contribution of additional genetic lesions to MPN disease phenotypes in JAK2-V617F positive patients. We also generated iPSC lines from MPN patients with the CALR type II (+5 bp) mutation and the rare MPL-V501L mutation. We found that the iPSC line containing a heterozygous MPL-V501L mutation (derived from an ET patient) appeared normal with iPSC typical morphology and phenotypes (expressing OCT4 and TRA-1-60 pluripotency markers) as the wildtype and JAK2-V617F iPSCs. We also generated four iPSC lines from a MF patient: all the four clones contained the homozygous CALR type II (+5 bp) mutation as found in the blood cells of the MF patient. Two iPSC clones we examined had a normal karyoptype. However, the iPSC lines showed signs of increased spontaneous differentiation when being cultured under the standard culture condition, based on cell morphology and reduction of OCT4 & TRA-1-60 expression. We are currently testing the potential of these iPSC lines containing the CALR and MPLmutations in hematopoietic differentiation, focusing on erythrocytic and megakaryocytic differentiation activities. Conclusions: we believe that by comparative analysis of isogenic iPSCs containing the common and rare MPN driver mutations, both common and specific pathways essential for MPNs can be determined. Precise genome editing or surgery of these mutations by engineered targeting may also provide a novel approach to eliminate the malignant MPN clones. This project is supported in part by the MPN Research Foundation and LLS. Disclosures No relevant conflicts of interest to declare.