A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in a majority of patients with polycythemia vera (PV), essential thrombocythemia, and primary myelofibrosis. However, contribution of the JAK2V617F mutation in these 3 clinically distinct myeloproliferative neoplasms (MPNs) remained unclear. To investigate the role of JAK2V617F in the pathogenesis of these MPNs, we generated an inducible Jak2V617F knock-in mouse, in which the expression of Jak2V617F is under control of the endogenous Jak2 promoter. Expression of heterozygous mouse Jak2V617F evoked all major features of human polycythemia vera (PV), which included marked increase in hemoglobin and hematocrit, increased red blood cells, leukocytosis, thrombocytosis, splenomegaly, reduced serum erythropoietin (Epo) levels and Epo-independent erythroid colonies. Homozygous Jak2V617F expression also resulted in a PV-like disease associated with significantly greater reticulocytosis, leukocytosis, neutrophilia and thrombocytosis, marked expansion of erythroid progenitors and Epo-independent erythroid colonies, larger spleen size, and accelerated bone marrow fibrosis compared with heterozygous Jak2V617F expression.

Key Points Loss of Ezh2 inhibits erythropoiesis but increases megakaryopoiesis in Jak2V617F knock-in mice. Loss of Ezh2 induces rapid progression to myelofibrosis in mice expressing Jak2V617F.

437 A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in a majority of patients with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). However, the contribution of JAK2V617F in these three clinically distinct myeloproliferative disorders (MPDs) remained unclear. To investigate the actual role of JAK2V617F in the pathogenesis of MPDs, we generated an inducible Jak2V617F knock-in mouse, in which the expression of Jak2V617F is under control of the endogenous Jak2 promoter. Expression of heterozygous Jak2V617F evoked all major features of human PV, which included marked increase in hemoglobin and hematocrit, increased red blood cells, leukocytosis, thrombocytosis, splenomegaly, reduced serum levels of erythropoietin (Epo) and Epo-independent erythroid colonies. Homozygous Jak2V617F expression resulted in a more severe form of PV associated with markedly elevated leukocytosis, neutrophilia and thrombocytosis, and a majority of these mice succumbed due to massive cardiac thrombosis. Activation of Stat5, Akt and Erk was significantly enhanced in erythroblast cells expressing homozygous Jak2V617F compared to heterozygous Jak2V617F, suggesting that the degree of activation of downstream signaling pathways would be affected by the Jak2V617F gene dosage. This may also partly explain the severe phenotype in mice expressing homozygous Jak2V617F. We conclude that heterozygous Jak2V617F is sufficient to cause PV, whereas homozygous Jak2V617F increases the severity of PV disease and the risk of thrombosis. Our results also provide strong evidence that Jak2V617F gene dosage does not play a defining role in determining PV versus ET phenotype in this mouse model of MPD. Disclosures: No relevant conflicts of interest to declare.

Jak2, a member of the Janus kinase family of non-receptor protein tyrosine kinases, is activated in response to a variety of cytokines, and functions in survival and proliferation of cells. An activating JAK2V617F mutation has been found in most patients with myeloproliferative neoplasms, and patients treated with Jak2 inhibitors show significant hematopoietic toxicities. However, the role of Jak2 in adult hematopoietic stem cells (HSCs) has not been clearly elucidated. Using a conditional Jak2 knockout allele, we have found that Jak2 deletion results in rapid loss of HSCs/progenitors leading to bone marrow failure and early lethality in adult mice. Jak2 deficiency causes marked impairment in HSC function, and the mutant HSCs are severely defective in reconstituting hematopoiesis in recipient animals. Jak2 deficiency also causes significant apoptosis and loss of quiescence in HSC-enriched LSK (Lin−Sca-1+c-kit+) cells. Jak2-deficient LSK cells exhibit elevated reactive oxygen species levels and enhanced p38 MAPK activation. Mutant LSK cells also show defective Stat5, Erk and Akt activation in response to thrombopoietin and stem cell factor. Gene expression analysis reveals significant downregulation of genes related to HSC quiescence and self-renewal in Jak2-deficient LSK cells. These data suggest that Jak2 plays a critical role in the maintenance and function of adult HSCs.

Promoting the expansion of adult stem cell populations offers the potential to ameliorate radiation or chemotherapy-induced bone marrow failure and allows for expedited recovery for patients undergoing these therapies. Previous genetic studies suggested a pivotal role for SH2 domain-containing inositol-5-phosphatase 1 (SHIP1) in limiting the size of the hematopoietic stem cell (HSC) compartment. The aim of this study was to determine whether our recent development of small molecule SHIP1 inhibitors offers the potential for pharmacological expansion of the HSC compartment in vivo. We show here that treatment of mice with aminosteroid inhibitors of SHIP1 (SHIPi) more than doubles the size of the adult mesenchymal stem cell (MSC) compartment while simultaneously expanding the HSC pool sixfold. Consistent with its ability to target SHIP1 function in vivo, SHIPi also significantly increases plasma granulocyte colony-stimulating factor (G-CSF) levels, a growth factor that supports proliferation of HSC. Here, we show that SHIPi-induced G-CSF production mediates HSC and MSC expansion, as in vivo neutralization of G-CSF abrogates the SHIPi-induced expansion of both the HSC and MSC compartments. Due to its expansionary effect on adult stem cell compartments, SHIPi represents a potential novel strategy to improve declining stem cell function in both therapy induced and genetically derived bone marrow failure syndromes. STEM CELLS 2015;33:848-858