Abstract 2902

Poster Board II-878

Myeloproliferative neoplasms (MPNs) are frequent malignant hematopoietic pathologies. The acquired Jak2V617F mutation is found in the majority of polycythemia vera (PV), and in approximately half of essential thrombocytosis (ET) and primary myelofibrosis (PMF). Animal models using transgenic or retroviral bone marrow transplant approaches allowing for JAK2V617F expression in hematopoietic cells have demonstrated that this sole mutation is sufficient to induce a MPN in mice. They have also shown that the level of JAK2V617F expression is crucial for MPN phenotypes. Therefore, these over-expression models appeared not suited to study human MPNs. In order to circumvent this problem, we developed a Jak2V617F constitutive and a conditional Knock-in (KI) mouse models. Jak2V617F allele was introduced into the endogenous Jak2 locus, allowing physiological expression of the mutated kinase. The Jak2V617F heterozygocity status was confirmed by allele-specific fluorescent competitive probes in quantitative real-time polymerase chain reaction. Constitutively activated JAKV617F kinase activity was assessed by analyzing STAT5a phosphorylation level in bone marrow and spleen cells by Western blotting.

Expression of endogenous JAK2V617F in constitutive KI mice induced a severe PV- to PMF-like phenotype disease, at 5 months of age, characterized in blood by marked polycythemia (Hct 71% ± 3.6%), granulocytosis (WBC counts 79 ± 11 × 106 cells/mL) and thrombocytosis (4.4 ± 0.7 × 109 platelets/mL). Mice displayed enlarged spleens (1.6 ± 0.3 g compared to 0.12 ± 0.01 g for WT mice). Histological examinations of the spleens and femurs revealed advanced degree of fibrosis and trilineage hyperplasia. Flow cytometry analysis showed high levels of TER-119-positive (erythroid) cells in bone marrow and spleen and a marked decrease in lymphocyte percents. The number of CFU-E increased in spleen and included an extremely high percentage of endogenous erythroid colonies (EECs). EECs were also detected in bone marrow. The numbers of BFU-E and CFU-GM were only slightly increased in the spleen but there were no significantly changed in the bone marrow. A major drawback of this constitutive and ubiquitous KI mouse model resides in the inability of female to reproduce even after recovery of normal blood parameters induced by hydroxyurea treatment. Progeny was only obtained through KI male breeding.

Embryonic expression of endogenous JAK2V617F was also obtained by crossing Jak2V617F conditional KI mice with CMV-Cre transgenic mice. The offspring presented a similar phenotype than the constitutive KI mice. We performed serial competitive bone marrow (BM) transplantations (BMTs) in irradiated WT recipient mice using from 10% to 100% CMV-Cre Jak2V617F KI mouse BM cells diluted with WT mouse BM cells. Time of disease onset and survival were shorter in mice grafted with high compared to low percentages of KI-derived cells. Reduced occurrences of disease were observed along with successive BMTs or limiting dilution of Jak2V617F-positive clone, suggesting that Jak2V617F provides little or no selective advantage to HSC.

This study demonstrates that embryonic endogenous JAK2V617F heterozygous expression results in a severe lethal MPN. This result may explain why no germ line transmission of the Jak2V617F-positive human disease has been reported.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.

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