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Acquired mutations of JAK2and TET2are 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.

The activating mutations in JAK2 (including JAK2V617F) that have been described in patients with myeloproliferative neoplasms (MPNs) are linked directly to MPN pathogenesis. We developed R723, an orally bioavailable small molecule that inhibits JAK2 activity in vitro by 50% at a concentration of 2nM, while having minimal effects on JAK3, TYK2, and JAK1 activity. R723 inhibited cytokine-independent CFU-E growth and constitutive activation of STAT5 in primary hematopoietic cells expressing JAK2V617F. In an anemia mouse model induced by phenylhydrazine, R723 inhibited erythropoiesis. In a leukemia mouse model using Ba/F3 cells expressing JAK2V617F, R723 treatment prolonged survival and decreased tumor burden. In V617F-transgenic mice that closely mimic human primary myelofibrosis, R723 treatment improved survival, hepatosplenomegaly, leukocytosis, and thrombocytosis. R723 preferentially targeted the JAK2-dependent pathway rather than the JAK1- and JAK3-dependent pathways in vivo, and its effects on T and B lymphocytes were mild compared with its effects on myeloid cells. Our preclinical data indicate that R723 has a favorable safety profile and the potential to become an efficacious treatment for patients with JAK2V617F-positive MPNs.

No relevant conflicts of interest to declare.

In primary myelofibrosis patients, somatic mutations such as JAK2V617F(JAKVF) and MPLW515 that activate JAK-STAT signaling are often seen. Small-molecule JAK2 inhibitors are effective for organomegaly and constitutional symptoms, but the drugs have little effect on BM fibrosis. To clarify the mechanism by which MPN cells with JAK2mutations cause BM fibrosis, we compared the gene expression patterns of Lin−Sca1+BM cells in JAK2VF transgenic mice (JAK2VF-TG), which develop myelofibrosis (MF), with that in WT mice. We found that TGFb1and HOXB4, the target genes of transcription factor USF1 were highly expressed. TGFβ1, which is secreted by hematopoietic cells, is essential for fibrotic development in a murine model of MF (Chagraoui et al. Blood 2002), and increased expression of HOXB4 enhances human megakaryocytic development (Zhong et al. BBRC 2010).

No relevant conflicts of interest to declare.

Abstract 2471

No relevant conflicts of interest to declare.

Nakaya: Nippon Shinyaku Co., Ltd: Employment. Homan:Nippon Shinyaku Co., Ltd: Employment. Kotera:Nippon Shinyaku Co., Ltd: Employment. Shibayama:Nippon Shinyaku Co., Ltd: Employment. Naito:Nippon Shinyaku Co., Ltd: Employment. Shimoda:Nippon Shinyaku Co., Ltd: Research Funding.

Abstract 4106

Abstract 2585

Janus kinase 1 (JAK1) plays a critical role in lymphocyte proliferation and differentiation. Somatic JAK1 mutations are found in 18% of adult precursor T acute lymphoblastic leukemias (T-ALL). Some of the mutations were shown to induce the phosphorylation of JAK1 and STAT5 and lead to cytokine-independent proliferation. These data suggest that dysregulation of JAK1 can be involved in the development or progression of T-ALL (Flex et al. J Exp Med. 2008;205:751-758). Adult T-cell leukemia/lymphoma (ATLL) is a type of T-cell neoplasm, and the activation of JAK/STAT is sometimes observed in the tumor cells. Therefore, we investigated JAK1 mutations in ATLL patients.Twenty Japanese ATLL patients whose percentage of peripheral abnormal lymphocytes was greater than 30% total cell count were sequentially enrolled into the study from 2000 to 2007. Diagnosis of ATLL was made on the basis of clinical features and laboratory characteristics. All cases tested positive for the serum anti-HTLV-1 antibody. The diagnosis was confirmed by observing monoclonal insertion of the HTLV-1 viral genome into leukemia cells by Southern blot hybridization. Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved at -80°C. These PBMCs were thawed and genomic DNA was isolated using standard protocol. The entire coding sequence of the JAK1 gene (exons 2 through 25) was amplified by the polymerase chain reaction (PCR) method. The sequence of PCR primers were kindly provided by Dr. Marco Tartaglia (Istituto Superiore di Sanità, Roma, PhD). The nucleotide sequences were determined by fluorescent dye chemistry sequencing and analyzed by sequencing analysis software. By referencing the assembled sequence in the Ensembl genome database, the presence of homozygous mutations was first checked and then candidates for heterozygous mutations or single nucleotide polypeptides (SNPs) on each allele were screened by comparing the ratio of different bases calculated with the height of the peaks seen from sequencing to the reference genome when the ratio was between 0.15 and 1.0.The percentage of abnormal lymphocytes ranged from 30-90%, and the mean value was 55.4%. The mean value of WBC and lymphocyte number was 40.5×109/L and 33.4×109/L, respectively. The mean value of LDH, Ca2+ or sIL-2R was 609 IU/L, 11.4 mg/dL, or 54748 U/mL, respectively. According to Shimoyama criteria (Shimoyama et al. Br J Haematol. 1991;79:428-437), 19 cases were diagnosed as acute-type ATLL, and one case was diagnosed as chronic-type ATLL. The surface markers of all but one abnormal PBMC were CD3+CD4+CD8-CD25+. In that one exception, loss of CD4 expression was observed. We examined the entire coding sequence of the JAK1 gene in 20 ATLL patients and identified no nonsynonymous or nonsense mutations and five types of silent substitutions in 12 cases. All silent substitutions were synonymous SNPs, as determined from referencing the base sequence in the Ensembl genome database. In the ATLL patients examined, the genotype frequency (%) is c546-AA/AG/GG, 97.5/2.5/0; c1590-CC/CT/TT, 97.5/2.5/0; c2049-CC/CT/TT, 50/50/0; c2097-CC/CG/GG, 95/5/0; c2199-AA/AG/GG, 60/40/0. There is no statistical difference in genotype frequency pattern of these SNPs, between the Japanese ATLL patients examined and the general Asian population on the Ensembl database.Mutations in the coding region of JAK1 do not associate with either activation of the JAK/STAT pathway or leukemogenesis in ATLL. We only examined the coding region of JAK1, and the regulatory region of JAK1 remains to be investigated. Further investigation including downstream signaling molecules and inhibitory molecules in the JAK/STAT signaling pathway is necessary to clarify the mechanism contributing to the leukemogenesis of ATLL.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 1901

Markovtsov: Rigel Pharmaceuticals,Inc.: Employment. Bhamidipati:Rigel Pharmaceuticals,Inc.: Employment. Park:Rigel Pharmaceuticals,Inc.: Employment. Torneros:Rigel Pharmaceuticals,Inc.: Employment. Duan:Rigel Pharmaceuticals,Inc.: Employment. Hitoshi:Rigel Pharmaceuticals,Inc.: Employment. Shimoda:Rigel Pharmaceuticals,Inc.: Research Funding.

Abstract 3897

Abstract 1921

Leukocyte alkaline phosphatase (LAP) enzymatic activity is a marker of the last stages of myeloid differentiation. The level of LAP is quantitated as the LAP score. Estimation of the LAP score has been useful for distinguishing chronic myelogenous leukemia (CML) from BCR-ABL–negative chronic myeloproliferative disorders (MPDs) and neutrophilic reactions in severe infections. CML patients usually have a low LAP score, whereas elevated LAP scores are seen in patients with polycythemia vera (PV), primary myelofibrosis (PMF), and leukocytosis caused by infections. An acquired Jak2 V617F mutation is seen in approximately 95% of patients with PV and in about 50% of patients with essential thrombocythemia or PMF. It has been shown that Jak2 V617F mutation induced constitutive activation of the JAK-STAT signaling pathway. We speculated that an elevated LAP score might be caused due to activation of JAK-STAT signaling through a Jak2 V617F mutation, and conducted this study to address this question. We analyzed the LAP scores in Jak2 V617F-positive and -negative MPD patients. Jak2 V617F-positive MPD patients had higher LAP scores than Jak2 V617F-negative patients. Moreover, patients carrying homozygous mutations had higher LAP scores than patients with heterozygous mutations. AG490, the Jak2 inhibitor, was shown to significantly decrease the LAP expression in neutrophils of Jak2 V617F-positive patients. We lentivirally transfected the acute promyelocytic leukemia cell line NB4 with the Jak2 V617F mutation and wild-type Jak2 V617F. The expression level of Jak2 was not significantly different between the Jak2 V617F mutation and wild-type Jak2 V617F. We then examined the LAP scores of transfected NB4 cells after these cells were differentiated by all-trans retinoic acid and granulocyte colony stimulating factor. It was observed that the Jak2 V617F mutation and not the wild-type Jak2 induced elevated LAP scores. Furthermore, we showed that Jak2 followed the MAP kinase pathway and not the PI3 kinase pathway, as a downstream signaling pathway to elevate the LAP scores using MEK 1/2 (U0126) and PI3 kinase (LY294002) inhibitors. In conclusion, we obtained direct evidence that Jak2 V617F mutation induces elevated LAP scores via the MAP kinase pathway.

Leukocyte alkaline phosphatase (LAP) enzymatic activity is a marker of the last stages of myeloid differentiation. The level of LAP is quantitated as the LAP score. Estimation of the LAP score has been useful for distinguishing chronic myelogenous leukemia (CML) from BCR-ABL–negative chronic myeloproliferative disorders (MPDs) and neutrophilic reactions in severe infections. CML patients usually have a low LAP score, whereas elevated LAP scores are seen in patients with polycythemia vera (PV), primary myelofibrosis (PMF), and leukocytosis caused by infections. An acquired Jak2 V617F mutation is seen in approximately 95% of patients with PV and in about 50% of patients with essential thrombocythemia or PMF. It has been shown that Jak2 V617F mutation induced constitutive activation of the JAK-STAT signaling pathway. We speculated that an elevated LAP score might be caused due to activation of JAK-STAT signaling through a Jak2 V617F mutation, and conducted this study to address this question. We analyzed the LAP scores in Jak2 V617F-positive and -negative MPD patients. Jak2 V617F-positive MPD patients had higher LAP scores than Jak2 V617F-negative patients. Moreover, patients carrying homozygous mutations had higher LAP scores than patients with heterozygous mutations. AG490, the Jak2 inhibitor, was shown to significantly decrease the LAP expression in neutrophils of Jak2 V617F-positive patients. We lentivirally transfected the acute promyelocytic leukemia cell line NB4 with the Jak2 V617F mutation and wild-type Jak2 V617F. The expression level of Jak2 was not significantly different between the Jak2 V617F mutation and wild-type Jak2 V617F. We then examined the LAP scores of transfected NB4 cells after these cells were differentiated by all-transretinoic acid and granulocyte colony stimulating factor. It was observed that the Jak2 V617F mutation and not the wild-type Jak2 induced elevated LAP scores. Furthermore, we showed that Jak2 followed the MAP kinase pathway and not the PI3 kinase pathway, as a downstream signaling pathway to elevate the LAP scores using MEK 1/2 (U0126) and PI3 kinase (LY294002) inhibitors. In conclusion, we obtained direct evidence that Jak2 V617F mutation induces elevated LAP scores via the MAP kinase pathway.

An acquired JAK2 V617F mutation has been detected in up to 90% of patients with polycythemia vera (PV) and in a sizeable proportion of patients with other myeloproliferative disorders such as essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). However, how a single mutation may be responsible for such different clinical phenotypes is unknown. Mice transplanted with bone marrow cells in which V617F JAK2 was retrovirally expressed developed PV-like features, but not ET or IMF. To address the contribution of this mutation to the pathogenesis of these three MPDs, we generated transgenic mice expressing V617F JAK2 driven by the murine H2Kb promoter. We established two lines. The expression of V617F JAK2 mRNA in bone marrow cells was 0.45 and 1.35 that of endogenous wild-type JAK2 in the two lines. One line showed leukocytosis after 4 months of age, with a predominance of granulocytes. Among 43 mice, examined after 3 months of age, 8 (19%) showed polycythemia and 14 (33%) showed thrombocythemia. Two polycythemia cases also showed thromobocytosis. The other line showed extreme leukocytosis and thromobocytosis at one month of age. The leukocytosis progressed as the animals aged, but the thrombocytosis tended to resolve at 8 months. They showed anemia that means Hb value from 9 to 10 g/dL at one month old. Myeloid cells and megakaryocytes were predominant in the bone marrow of these animals, and splenomegaly with myeloid cell and megakaryocyte invasion was observed. We conclude that in vivo expression of V617F JAK2 results in ET-like, IMF-like, and PV-like disease.

An acquired JAK2 V617F mutation has been detected in up to 90% of patients with polycythemia vera (PV) and in a sizeable proportion of patients with other myeloproliferative disorders such as essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). However, how a single mutation may be responsible for such different clinical phenotypes is unknown. Mice transplanted with bone marrow cells in which V617F JAK2 was retrovirally expressed developed PV-like features, but not ET or IMF. To address the contribution of this mutation to the pathogenesis of these three MPDs, we generated transgenic mice expressing V617F JAK2 driven by the murine H2Kbpromoter. We established two lines. The expression of V617F JAK2 mRNA in bone marrow cells was 0.45 and 1.35 that of endogenous wild-type JAK2 in the two lines. One line showed leukocytosis after 4 months of age, with a predominance of granulocytes. Among 43 mice, examined after 3 months of age, 8 (19%) showed polycythemia and 14 (33%) showed thrombocythemia. Two polycythemia cases also showed thromobocytosis. The other line showed extreme leukocytosis and thromobocytosis at one month of age. The leukocytosis progressed as the animals aged, but the thrombocytosis tended to resolve at 8 months. They showed anemia that means Hb value from 9 to 10 g/dL at one month old. Myeloid cells and megakaryocytes were predominant in the bone marrow of these animals, and splenomegaly with myeloid cell and megakaryocyte invasion was observed. We conclude that in vivoexpression of V617F JAK2 results in ET-like, IMF-like, and PV-like disease.