Expression of JAK2^V617I Does Not Result in Overt Myeloproliferative Disorder, but Results in Cytokine Hypersensitivity

BACKGROUND:

A germline JAK2^V617I mutation has recently been identified in a family with hereditary thrombocytosis (Mead et al, NEJM 2012). Like acquired MPN, family members with JAK2^V617I have thrombocytosis and megakaryocytic hyperplasia in the marrow with increased risk of thrombosis. But unlike acquired MPN, individuals with this germline mutation do not develop a fibrotic bone marrow, splenomegaly, or transform to acute leukemia. Why germline JAK2^V617I recapitulates some aspects but not others of the MPN phenotype in humans is unclear. To delineate the differences between JAK2^V617F and JAK2^V617I we compared the phenotype of mice with hematopoietic cells expressing JAK2^V617F or JAK2^V617I.

METHODS AND RESULTS:

Lethally irradiated C57B/6 mice were transplanted with bone marrow cells infected with retrovirus expressing JAK2^V617F, JAK2^V617I, or empty MSCV-IRES-GFP (MIG) vector. As expected, mice transplanted with JAK2^V617F-expressing cells developed erythrocytosis and leukocytosis, whereas mice transplanted with JAK2^V617I-expressing cells had peripheral blood counts similar to empty vector mice. Humans with germline JAK2^V617I do not display constitutive activation of the kinase, but they do demonstrate cytokine hyper-responsiveness as evidenced by increased phosphorylation of STATs at low concentrations of ligand. We compared phosphorylated STAT5 in peripheral blood of mice transplanted with JAK2^V617I, JAK2^V617F, and MIG empty vector following stimulation with increasing concentrations of GM-CSF. At all concentrations of GM-CSF tested JAK2^V617I and JAK2^V617F-expressing cells had exaggerated phosphorylation of STAT5 as compared to MIG empty vector mice. We also measured phospho-STAT3 and STAT5 in unstimulated bone marrow and spleen from each mouse at time of euthanasia, there was no difference between JAK2^V617I and MIG empty vector mice. JAK2^V617F mice did demonstrate phosphorylation of STAT3 and STAT5 in the absence of GM-CSF, confirming the ability of JAK2^V617F but not JAK2^V617I to constitutively activate downstream signaling pathways. Next, to evaluate for histologic evidence of MPN and assess spleen size, all mice were euthanized at 120 days post-transplant. JAK2^V617F mice had splenomegaly as expected, spleens from JAK2^V617I mice appeared larger than empty vector mice, but spleen weight was not statistically different (p>0.05). While JAK^V617I mice had increased cellularity of their marrow with increased numbers of megakaryocytes as compared to empty vector mice, this was not nearly to the extent of JAK2^V617F mice. Mild fibrosis was seen in JAK2^V617I mice, JAK2^V617F mice had severe reticulin fibrosis in the marrow as expected. In the spleen architecture was preserved in the JAK2^V617I mice, whereas in the JAK2^V617F mice splenic architecture was disrupted by invasion of myeloid cells including megakaryocytes.

To identify whether JAK2^V617I affects the frequency of stem and progenitor cells or expands mature myeloid lineage cells we measured the frequency of hematopoietic stem cells, myeloid progenitors, and mature myeloid populations in JAK2^V617F, JAK2^V617I, and MIG empty vector. The bone marrow of JAK2^V617I mice contained an increased percentage of GMP and MEP populations as compared to both the MIG empty vector and the JAK2^V617F mice. Mature granulocyte (Gr-1⁺CD11b⁺) and erythroid (Ter119⁺) populations were expanded in the bone marrow and spleen of JAK2^V617F but not JAK2^V617I mice.

CONCLUSIONS:

These data demonstrate that the JAK2^V617I mouse model recapitulates the effect of germline expression of JAK2^V617I seen in humans: it results in cytokine hyper-responsiveness without the ability to constitutively activate downstream signals in the absence of ligand. Why JAK2^V617F is so exquisitely conserved in acquired MPN is still unknown.