JAK2-V617F-Driven Myeloproliferative Neoplasms (MPN) Are Initiated By a Subset of HSCs Marked By High Expression of the Vwf-GFP Reporter Gene

Introduction. Myeloproliferative neoplasms (MPN) are clonal diseases that originate from a single hematopoietic stem cell (HSC). The JAK2-V617F mutation is found in ~70% of all MPN patients. To date, it remains unclear why some patients with this JAK2-V617F mutation develop essential thrombocythemia (ET), with hyperproliferation of the megakaryocyte lineage, while others develop polycythemia vera (PV), characterized by an overproduction of both platelets and erythrocytes. In this project, we explored the hypothesis that acquiring the JAK2-V617F mutation in a subset of HSCs that are biased towards the megakaryocyte lineage will lead to ET, whereas acquiring the mutation in unbiased HSCs will lead to PV.

Methods. To identify megakaryocyte-biased HSCs, we used a mouse reporter line that expresses the green fluorescent protein (GFP) under the promoter of the von Willebrand Factor (vWF). This reporter allows for the identification of a subset of HSCs with high GFP expression (vWF-GFP^hi HSCs) which, in wildtype (WT) mice, showed bias towards megakaryocyte and platelet production in vivo (Sanjuan-Pla et al., Nature 2013). We crossed this vWF-GFP reporter with our SclCre^ER;JAK2-V617F MPN mice (VF), which display marked erythrocytosis and thrombocytosis after activation of the transgene by tamoxifen (Kubovcakova et al., Blood 2013). To compare the in vivo functional potential of FACS-sorted vWF-GFP^lo and vWF-GFP^hi HSCs isolated from WT or VF donor mice, we performed competitive transplantations of 100 HSCs (defined as Lin^-c-Kit⁺Sca-1⁺CD48^-CD150⁺Epcr⁺) mixed with 1x10⁶WT bone marrow competitor cells into lethally irradiated WT recipients.

Results.Recipient mice transplanted with vWF-GFP^hi HSCs from WT donors exhibited higher donor chimerism in platelets than in other peripheral blood lineages, while recipients of vWF-GFP^lo HSCs showed no megakaryocyte bias, as previously reported (Sanjuan-Pla et al., Nature 2013). Mice transplanted with vWF-GFP^hi HSCs from VF donors developed PV phenotype but surprisingly lost their megakaryocyte bias, whereas recipients of vWF-GFP^lo HSCs had low overall chimerism without MPN phenotype and no platelet bias. Thus, in the presence of the JAK2-V617F mutation, only the vWF-GFP^hi HSCs were capable of initiating MPN, while losing their preference for the megakaryocyte lineage. In both VF and WT mice, we also found that 40% of vWF-GFP^hi HSCs showed high surface expression of the integrin CD41 (CD41^hi), which was reported to be associated with megakaryocyte-biased HSCs (Gekas and Graf, Blood 2013), while 60% were CD41^lo. Mice transplanted with vWF-GFP^hi /CD41^lo HSCs showed better engraftment compared to recipients of vWF-GFP^hi /CD41^hi HSCs, but neither fraction showed bias towards megakaryocyte and platelet production.

Conclusion.Only the vWF-GFP^hi subset of HSCs from VF mice was capable of initiating MPN phenotype in competitive stem cell transplantations into irradiated WT recipient mice, irrespective of the CD41 marker expression. Furthermore, expression of JAK2-V617F abolished megakaryocyte and platelet bias of the vWF-GFP^hi subset of HSCs that was observed in WT mice. Additional investigations are underway to dissect the contribution of vWF-GFP^lo versus vWF-GFP^hi HSCs in our VavCre;JAK2-V617F mice that develop an ET-like phenotype with high platelets but normal red cell parameters.

Skoda:Ajax Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; AOP: Honoraria, Speakers Bureau; Bristol Myers Squibb/Celgene: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Baxalta: Consultancy, Honoraria; Pfizer: Consultancy.