An acquired JAK2 V617F mutation is found in most patients with polycythemia vera (PV), and about half of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). Mice transplanted with bone marrow cells in which JAK2 V617F was retrovirally expressed developed PV-like features, but not ET or PMF. To address the contribution of this mutation to the pathogenesis of these three MPDs, we generated two lines of JAK2 V617F transgenic mice. One line showed granulocytosis after 4 months of age. Among 43 mice, 8 (19%) showed polycythemia and 15 (35%) showed thrombocythemia. The second line showed extreme leukocytosis and thromobocytosis. They showed anemia that means Hb value from 9 to 10 g per 100 ml when 1 month old. Myeloid cells and megakaryocytes were predominant in the bone marrow of these animals, and splenomegaly was observed. The expression of JAK2 V617F mRNA in bone marrow cells was 0.45 and 1.35 that of endogenous wild-type JAK2 in the two lines, respectively. In vitro analysis of bone marrow cells from both lines showed constitutive activation of ERK1/2, STAT5 and AKT, and augmentation of their phosphorylations by cytokine stimulation. We conclude that in vivo expression of JAK2 V617F results in ET-, PMF-and PV-like disease.

Hepatocyte growth factor (HGF) modulates intestinal epithelial cell proliferation and migration, serving as a critical regulator of intestinal wound healing. In this study, we examined the effect of administration of recombinant human HGF on colonic mucosal damage in vivo. Acute colitis was induced in rats by feeding with 5% dextran sulfate sodium (DSS) for 7 days, and colitis was subsequently maintained by feeding with 1% DSS. On the 5th day of DSS administration, osmotic pumps releasing recombinant human HGF (200 g/day) were implanted into the peritoneum of the rats. Continuous intraperitoneal delivery of HGF led to both increased serum human HGF levels and c-Met tyrosine phosphorylation within the colonic mucosa. Compared with mock-treated rats, those administered human HGF showed a reduction in colitis-associated weight loss, large intestinal shortening, and improved colonic erosions. Enhanced epithelial regeneration and cellular proliferation were observed in rats treated with recombinant human HGF. The weights of the liver, kidneys, and spleen were not affected by HGF administration. These results indicate that HGF administration accelerates colonic mucosal repair in rats with DSS-induced colitis and suggest that recombinant human HGF may be a useful therapeutic tool to facilitate intestinal wound healing in patients with ulcerative colitis.

Mutations of calreticulin (CALR) are detected in 25–30% of patients with essential thrombocythemia (ET) or primary myelofibrosis and cause frameshifts that result in proteins with a novel C-terminal. We demonstrate that CALR mutations activated signal transducer and activator of transcription 5 (STAT5) in 293T cells in the presence of thrombopoietin receptor (MPL). Human megakaryocytic CMK11-5 cells and erythroleukemic F-36P-MPL cells with knocked-in CALR mutations showed increased growth and acquisition of cytokine-independent growth, respectively, accompanied by STAT5 phosphorylation. Transgenic mice expressing a human CALR mutation with a 52 bp deletion (CALRdel52-transgenic mice (TG)) developed ET, with an increase in platelet count, but not hemoglobin level or white blood cell count, in association with an increase in bone marrow (BM) mature megakaryocytes. CALRdel52 BM cells did not drive away wild-type (WT) BM cells in in vivo competitive serial transplantation assays, suggesting that the self-renewal capacity of CALRdel52 hematopoietic stem cells (HSCs) was comparable to that of WT HSCs. Therapy with the Janus kinase (JAK) inhibitor ruxolitinib ameliorated the thrombocytosis in TG mice and attenuated the increase in number of BM megakaryocytes and HSCs. Taken together, our study provides a model showing that the C-terminal of mutant CALR activated JAK-STAT signaling specifically downstream of MPL and may have a central role in CALR-induced myeloproliferative neoplasms.

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Key Points• Loss of TET2 accelerates the degree of malignancy of MPNs in combination with JAK2V617F.• Loss of TET2 sustains MPNs in combination with JAK2V617F.Acquired mutations of JAK2 and TET2 are frequent in myeloproliferative neoplasms (MPNs). We examined the individual and cooperative effects of these mutations on MPN development. Recipients of JAK2V617F cells developed primary myelofibrosis-like features; the addition of loss of TET2 worsened this JAK2V617F-induced disease, causing prolonged leukocytosis, splenomegaly, extramedullary hematopoiesis, and modestly shorter survival. Double-mutant (JAK2V617F plus loss of TET2) myeloid cells were more likely to be in a proliferative state than JAK2V617F single-mutant myeloid cells. In a serial competitive transplantation assay, JAK2V617F cells resulted in decreased chimerism in the second recipients, which did not develop MPNs. In marked contrast, cooperation between JAK2V617F and loss of TET2 developed and maintained MPNs in the second recipients by compensating for impaired hematopoietic stem cell (HSC) functioning. In-vitro sequential colony formation assays also supported the observation that JAK2V617F did not maintain HSC functioning over the long-term, but concurrent loss of TET2 mutation restored it. Transcriptional profiling revealed that loss of TET2 affected the expression of many HSC signature genes. We conclude that loss of TET2 has two different roles in MPNs: disease accelerator and disease initiator and sustainer in combination with JAK2V617F. (Blood. 2015;125(2):304-315) IntroductionMyeloproliferative neoplasms (MPNs) are clinically characterized by the chronic overproduction of differentiated peripheral blood cells and a gradual expansion of malignant intramedullary/extramedullary hematopoiesis. Mutations in Janus kinase 2 (JAK2), 1-4 myeloproliferative leukemia protein, 5 and the recently reported calreticulin 6,7 are considered to play a "driver" role in MPNs. JAK2 mutations (eg, JAK2V617F and JAK2 exon 12 mutations) are the most frequent of these; almost all polycythemia vera (PV) patients and about half of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients have these mutations. [1][2][3][4]8,9 JAK2V617F mutations induce the phosphorylation of Janus kinases and signal transducers and activators of transcription (STATs) in cytokine-dependent cell lines without cytokine stimulation, and contribute to autonomous cell growth.1,4 JAK2V617F leads to MPNs in all transgenic [10][11][12] or knockin mouse 13-16 models, and is thought to play a central role in MPN development.In addition to the above driver mutations, mutations in epigenetic regulators such as TET2, DNMT3A, ASXL1, EZH2, and IDH1/2 have been reported. 17 Of these, TET2, a methylcytosine dioxygenase that converts 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC), [18][19][20] is mutated in chronic-phase MPNs, specifically in 10% to 17% of PV, 4% to 11% of ET, and 8% to 25% of PMF. [21][22][23] TET2 is also mutated in 13% to 32% of blastic-phase MPNs and is invol...