Modeling JAK2V617F Heterozygous and Homozygous Mutations by Using Induced Pluripotent Stem Cells Derived From Myeloproliferative Neoplasms Patients
Authors: Saliba Joseph , Hamidi Sofiane , Stefano Antonio Di , Langlois Thierry , Lenglet Gaëlle , Secardin Lise , Galy Anne , Opolon Paule , Debili Najet , Raslova Hana , Norol Françoise , Plo Isabelle , Vainchenker William 
Published: October 2019

Abstract 1758

During the last several years, iPS cells have been described as a new powerful tool for disease modeling and drug screening. It became possible to reprogram adult patients cells into specific pluripotent cell lines harboring inherited and/or acquired genetic abnormalities. We have focused our work on modeling Jak2V617F positive MPN patients in iPS cells.

By retroviral infection (OCT4, SOX2, KLF4, c-Myc), several iPS cell lines were generated from CD34+ cells of a healthy donor (control), a heterozygous JAK2 V617F patient and a homozygous JAK2 V617F patient. All cell lines expressed pluripotent surface markers (TRA1–81, SSEA4), pluripotent genes (NANOG, OCT4, SOX2) and were capable to induce teratomas in immunodeficient mice. Moreover, they were silenced for the expression of the 4 exogenous transgenes and showed no new clonal karyotypic abnormalities. All the iPS cell lines derived from the JAK2V617F heterozygous and homozygous patients were also heterozygous and homozygous for JAK2V617F. No JAK2 wild type iPS could be obtained from the patients CD34+ cells. Interestingly all the homozygous JAK2V617Fhad no 20q deletion by cytogenetic and CGH analysis demonstrating that this deletion was a secondary event in this MPN.

JAK2V617F mutation did not elicit a significant increase in hematopoiesis in comparison to the control iPS. However, marked differences in cytokine sensitivity were found. Homozygous iPS-derived erythroid progenitors had spontaneous growth, hypersensitivity to EPO and gave rise to slightly larger erythroid colonies in methylcellulose assays as compared to the control, whereas erythroid progenitors from JAK2V617F heterozygous iPS has similar Epo-response than those from the control. Interestingly, Megakaryocytes (MK) progenitors from JAK2V617F heterozygous iPS had hypersensitivity to TPO whereas those from homozygous iPS showed a complete TPO independence. Those cytokine-response profiles recapitulate the disease's primary features and validate the iPS as a good tool for JAK2V617F MPN modeling. MK differentiation showed a maturation defect with an excess of immature MKs only in the JAK2 V617Fhomozygous lines, who was derived with a post-PV myelofibrosis, suggesting the existence of intrinsic abnormalities of the MK lineage.

Finally, we tested several JAK2, PI3K and ERK inhibitors on the JAK2V617F-mediated erythroid growth and showed that only JAK2 and PI3K inhibitors were able to block the erythroid spontaneous growth.

iPS technology recapitulates the cytokine hypersensitivity of JAK2V617F MPNs with marked differences between heterozygous and homozygous mutations, enabling us to screen various pharmaceutical inhibitors. In an addition it can also be used to study the clonal hierarchy in a MPN and in the future to study the synergistic effects of different mutations.

Disclosures:

No relevant conflicts of interest to declare.