JAK2-V617F Expressing Stem Cells Display a Competitive Advantage At Low Limiting Dilution and Are Capable of Initiating MPN Phenotype
Authors: Lundberg Pontus , Kubovcakova Lucia , Takizawa Hitoshi , Hao-Shen Hui , Manz Markus G , Skoda Radek C. 
Published: October 2019

Abstract
Abstract 615
JAK2-V617F is the most common genetic alteration in myeloproliferative neoplasms (MPN). Forced expression of JAK2-V617F in animal models induced a MPN phenotype. However, MPN in these models was initiated by polyclonal expansion of multiple hematopoietic progenitor and stem cells. In contrast, patients with MPN display clonal hematopoiesis. Frequently, additional somatic mutations precede the acquisition of JAK2-V617F, suggesting that a pre-JAK2 mutation could be required for clonal expansion. Compatible with this hypothesis, in one JAK2-V617F knockin model the MPN phenotype was not transplantable and the stem cells showed signs of exhaustion. To address the question of whether JAK2-V617F alone is sufficient to initiate MPN we performed competitive repopulation assays and serial transplantations in a transgenic JAK2-V617F mouse model.
Our transgenic mice express the human JAK2-V617F under the endogenous JAK2 promoter and display a polycythemia vera (PV) or essential thrombocythemia (ET) phenotype. The transgene can be activated by Cre recombinase and the donors were induced 5–6 weeks prior to transplantation. To allow monitoring of the reconstitution, a GFP expressing transgenic strain was used as the source of competitor cells. Bone marrow transplantations were performed into lethally irradiated mice in the inbred BL/6 background. A total of 2×106 bone marrow cells per recipient were transplanted with ratios of mutant (V617F) to wild type (WT) set as 1:1, 1:10 or 1:100.

Results:
At 1:1 and 1:10 ratios, the mice displayed a PV phenotype beginning at 4 weeks post transplantation (Hb ∼200-220 g/L and neutrophil count of 5–20×109 /L) that remained stable for ∼26 weeks. An increase in platelet counts (4-6×1012/L) followed at later time points. V617F cells rapidly outcompeted WT cells reaching >95% chimerisms in peripheral blood. The MPN phenotype was transplantable into secondary and tertiary recipients and no signs of exhaustion were observed. An increase in the frequencies of lineage-negative, Sca-1+ c-kit+ (LSK) cell numbers in bone marrow was noted (4.1% in V617F vs 1.2% for WT, p<0.05). To assess their cycling rate, LSK cells were sorted, labeled with carboxyfluorescein succinimidyl ester and transplanted into non-conditioned recipients. After 8 weeks, the number of LSK cells was 62-fold increased and V617F LSK cells outcompeted host cells (52% vs. 3% for WT, p<0.01). The number of slow-dividing (0-2 divisions) LSK cells was substantially reduced in the V617F transplanted mice (0.095% vs. 7.54% for WT, p<0.01). Thus, V617F increases the cycling of LSK cells and provides a competitive advantage.
To allow detecting low numbers of donor cells in 1:100 transplantations, the GFP transgene was bred into the V617F background and a mixture of 2×104 V617F/GFP bone marrow cells plus 1.98×106 WT competitors was used. Based on the expected frequency of 3 long term hematopoietic stem cells (HSC) per 105 bone marrow cells, the recipients should receive on average 0.6 GFP-positive long term HSC. In mice transplanted with V617F/GFP and WT competitors at 1:100, we detected reconstitution defined as >0.1% GFP positive cells in 12 of 40 recipients (28%) that persisted beyond 20 weeks, whereas in mice transplanted with WT/GFP and WT competitors, only 5/40 (12%) showed reconstitution. The relative contribution in the WT group ranged from 0.1 – 3%, whereas in the V617F group we observed chimerism between 5 –100%. Four of the 12 mice that reconstituted with V617F/GFP cells developed a MPN phenotype (3PV, 1ET). Terminal workup of 3 mice (2PV and 1ET) at 29 weeks revealed that mice with erythrocytosis had enlarged spleens (∼150mg vs. 65mg for WT) and increased erythropoiesis in spleen histology. In contrast the mouse with ET phenotype showed only a slight increase in spleen size (90mg vs. 65mg for WT) and no increase in spleen erythropoiesis.

Conclusions:
Our results show that JAK2-V617F positive cells were able to rapidly outcompete WT cells. The MPN phenotype was transplantable into secondary and tertiary recipients. At low limiting dilution (1:100), V617F cells displayed > 2-fold higher engraftment rate (28%) than WT cells (12%) and a higher contribution to hematopoiesis (5-100% chimerism vs. 0.1–3%). Since 4/12 mice (30%) reconstituted with V617F cells also developed a MPN phenotype, our data demonstrate than JAK2-V617F as the sole genetic alteration can initiate MPN under conditions resembling monoclonal disease.

Disclosures:
No relevant conflicts of interest to declare.