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.

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...

Ten-Eleven-Translocation 2 (TET2) is an enzyme that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC) and thereby alters the epigenetic state of DNA; somatic loss-of-function mutations of TET2 are frequently observed in patients with diverse myeloid malignancies. To study the function of TET2 in vivo, we analyzed Ayu17-449 (TET2 trap ) mice, in which a gene trap insertion in intron 2 of TET2 reduces TET2 mRNA levels to about 20% of that found in wild-type (WT) mice. TET2 trap/trap mice were born at Mendelian frequency but died at a high rate by postnatal day 3, indicating the essential role of TET2 for survival. Loss of TET2 results in an increase in the number of hematopoietic stem cells (HSCs)/progenitors in the fetal liver, and TET2 trap/trapHSCs exhibit an increased self-renewal ability in vivo. In competitive transplantation assays, TET2 trap/trap HSCs possess a competitive growth advantage over WT HSCs. These data indicate that TET2 has a critical role in survival and HSC homeostasis. (2012) 26, 2216-2223; doi:10.1038/leu.2012.94 Keywords: TET2; myeloproliferative neoplasms; tumor suppressor; animal models; hematopoietic stem cells Leukemia INTRODUCTIONMyeloproliferative neoplasms (MPNs) are characterized by an overproduction of white blood cells and are frequently associated with a somatic Janus kinase 2 (JAK2) V617F mutation that results in a constitutively active protein. [1][2][3][4] The presence of JAK2 V617F alone can cause MPN in mouse models, 5-9 but does not confer a growth advantage upon hematopoietic stem cells (HSCs). 9In female MPN patients tested for JAK2 mutations and X-chromosomal clonality, the percentage of granulocytes and platelets with a JAK2 mutation was often markedly lower than the percentage of clonal granulocytes. 10,11 In addition, JAK2-V617F-positive MPNs are frequently found in patients with JAK2-V617F-negative acute myeloid leukemia. 12 These data suggest that the MPN cells expand clonally before acquisition of a JAK2 mutation, and that the leukemic transformation shares a common clonal origin in MPN and acute myeloid leukemia.Mutations in Ten-Eleven-Translocation-2 (TET2) have also been observed in 10-20% of patients with MPN,13,14 as well as in patients with myelodysplastic syndrome, chronic myelomonocytic leukemia and acute myeloid leukemia, [14][15][16][17] suggesting that mutation of TET2 is a common pathogenetic event in myeloid malignancies. TET family members share a highly conserved catalytic domain, and both TET1 and TET2 catalyze the conversion of 5-methylcytosine into 5-hmC, [18][19][20] resulting in the modification of DNA. The amount of 5-hmC in DNA from patients with myeloid malignancies was lower when they possessed a TET2 mutation than when they did not, 19 suggesting that the mutant TET2 protein was defective in its enzymatic function. Recent work has shown that small hairpin RNA-mediated depletion of TET2 in murine hematopoietic precursors alters their differentiation toward monocyte/macrophage lineages in vitro, suggesting...

Calreticulin ( CALR ) exon 9 frameshift mutations, commonly detected in essential thrombocythemia (ET) and primary myelofibrosis patients, activate signal transducer and activator of transcription (STAT) proteins in the presence of Myeloproliferative Leukemia Virus (MPL) and induce ET in vivo. Loss of the KDEL motif, an endoplasmic reticulum retention signal, and generation of many positively charged amino acids (AAs) in the mutated C-terminus are thought to be important for disease induction. To test this hypothesis, we generated mice harboring a Calr frameshift mutation using the CRISPR/Cas9 system. Deletion of 19-base pairs in exon 9 (c.1099-1117del), designated the del19 mutation, induced loss of the KDEL motif and generated many positively charged AAs, similar to human mutants. Calr del19 mice exhibited mild thrombocytosis, slightly increased megakaryocytes, and mild splenomegaly. In vitro experiments revealed that the murine CALR del19 mutant had a weaker ability to combine with murine MPL than the human CALR del52 mutant. Consequently, STAT5 activation was also very weak downstream of the murine mutant and murine MPL, and may be the reason for the mild disease severity. In summary, loss of the KDEL motif and positively charged AAs in the C-terminus of CALR is insufficient for MPL binding and ET development.

Loss-of-function of ten-eleven translocation-2 (TET2) is a common event in myeloid malignancies, and plays pleiotropic roles, including augmenting stem cell self-renewal and skewing hematopoietic cells to the myeloid lineage. TET2 mutation has also been reported in lymphoid malignancies; 5.7∼12% of diffuse large B-cell lymphomas and 18∼83% of angioimmunoblastic T-cell lymphomas had TET2 mutations. We investigated TET2 mutations in 22 adult T-cell leukemia/lymphoma (ATLL) patients and identified a missense mutation in 3 cases (14%). TET2 mutation occurred in a number of ATLL patients and was likely involved in their leukemogenesis.