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The acquired gain-of-function V617F mutation in the Janus Kinase 2 (JAK2V617F) is the main mutation involved in BCR/ABL-negative myeloproliferative neoplasms (MPNs), but its effect on hematopoietic stem cells as a driver of disease emergence has been questioned. Therefore, we reinvestigated the role of endogenous expression of JAK2V617Fon early steps of hematopoiesis as well as the effect of interferon-α (IFNα), which may target the JAK2V617Fclone in humans by using knock-in mice with conditional expression of JAK2V617Fin hematopoietic cells. These mice develop a MPN mimicking polycythemia vera with large amplification of myeloid mature and precursor cells, displaying erythroid endogenous growth and progressing to myelofibrosis. Interestingly, early hematopoietic compartments [Lin-, LSK, and SLAM (LSK/CD48−/CD150+)] increased with the age. Competitive repopulation assays demonstrated disease appearance and progressive overgrowth of myeloid, Lin-, LSK, and SLAM cells, but not lymphocytes, from a low number of engrafted JAK2V617FSLAM cells. Finally, IFNα treatment prevented disease development by specifically inhibiting JAK2V617Fcells at an early stage of differentiation and eradicating disease-initiating cells. This study shows that JAK2V617Fin mice amplifies not only late but also early hematopoietic cells, giving them a proliferative advantage through high cell cycling and low apoptosis that may sustain MPN emergence but is lost upon IFNα treatment.

The Jak2V617Fmutation is found in most classical BCR/ABL-negative myeloproliferative neoplasms (MPNs). Usually, heterozygosity of the mutation is associated with essential thrombocythemia (ET) and homozygosity with polycythemia vera (PV). Retrovirally transduced or transgenic animal models have shown that the mutation is sufficient for MPN development but that the level of expression is crucial for MPN phenotypes. Therefore we investigated the effect of an endogenous heterozygous expression of Jak2V617Fin knock-in (KI) mice. These animals displayed constitutive JAK2 activation and autonomous erythroid progenitor cell growth. Mice suffered from marked polycythemia, granulocytosis and thrombocytosis. Spleens and marrows displayed myeloid trilineage hyperplasia. Most animals survived to develop advanced fibrosis in these organs at around 9 months of age. In conclusion, constitutive heterozygous expression of JAK2V617Fin mice is not embryo-lethal but results in severe PV-like disease with secondary myelofibrosis and not in ET-like disease as expected from patient study.

A JAK2V617Fmutation is frequently found in several BCR/ABL-negative myeloproliferative disorders. To address the contribution of this mutant to the pathogenesis of these different myeloproliferative disorders, we used an adoptive transfer of marrow cells transduced with a retrovirus expressing JAK2V617Fin recipient irradiated mice. Hosts were analyzed during the 6 months after transplantation. For a period of 3 months, mice developed polycythemia, macrocytosis and usually peripheral blood granulocytosis. Transient thrombocytosis was only observed in a low-expresser group. All mice displayed trilineage hyperplasia in marrow and spleen along with an amplification of myeloid and erythroid progenitor cells and a formation of endogenous erythroid colonies. After 3 to 4 months, polycythemia regressed, abnormally shaped red blood cells and platelets were seen in circulation, and a deposition of reticulin fibers was observed in marrow and spleen. Development of fibrosis was associated with anemia, thrombocytopenia, high neutrophilia, and massive splenomegaly. These features mimic human polycythemia vera and its evolution toward myelofibrosis. This work demonstrates that JAK2V617Fis sufficient for polycythemia and fibrosis development and offers an in vivo model to assess novel therapeutic approaches for JAK2V617F-positive pathologies. Questions remain regarding the exact contribution of JAK2V617Fin other myeloproliferative disorders.

The cellular and molecular bases of platelet release by terminally differentiated megakaryocytes represent important questions in cell biology and hematopoiesis. Mice lacking the transcription factor NF-E2 show profound thrombocytopenia, and their megakaryocytes fail to produce proplatelets, the microtubule-based precursors of blood platelets. Using mRNA subtraction between normal and NF-E2–deficient megakaryocytes, cDNA was isolated encoding ß1 tubulin, the most divergent ß tubulin isoform. In NF-E2–deficient megakaryocytes, ß1 tubulin mRNA and protein are virtually absent. The expression of ß1 tubulin is exquisitely restricted to platelets and megakaryocytes, where it appears late in differentiation and localizes to microtubule shafts and coils within proplatelets. Restoring NF-E2 activity in a megakaryoblastic cell line or in NF-E2–deficient primary megakaryocytes rescues the expression of ß1 tubulin. Re-expressing ß1 tubulin in isolation does not, however, restore proplatelet formation in the defective megakaryocytes, indicating that other critical factors are required; indeed, other genes identified by mRNA subtraction also encode structural and regulatory components of the cytoskeleton. These findings provide critical mechanistic links between NF-E2, platelet formation, and selected microtubule proteins, and they also provide novel molecular insights into thrombopoiesis.

Background:The gain-of-function JAK2 V617Fmutant is the most common driver mutation identified in myeloproliferative neoplasms (MPNs). Additional somatic variants, also found in other malignant hemopathies, are detected in primary myelofibrosis (MF) and supposed to contribute to fibrosis or leukemia development. One of these mutations affects SRSF2, a gene encoding a component of the splicing machinery. SRSF2heterozygous mutation mainly affects the proline 95 residue of the protein. Its association with JAK2 V617Fcorrelates with a reduced leukemia free survival. Whether and how SRSF2 P95variants could favor fibrosis development in JAK2 V617Fcells remained unknown.

Colloidal gold was used as a marker for immunoelectron microscopy to localize lactoferrin (LF) and myeloperoxidase (MPO) in human peripheral blood neutrophils. Cells were reacted with monospecific antibodies against LF or MPO and then with gold-labeled antiglobulin. MPO cytochemistry was also associated with immunologic detection of LF. Immunologic labeling of thin sections after embedding in glycol methacrylate gave good ultrastructural morphology and specific localization of both proteins. MPO was detected in the large azurophil granules, whereas LF was consistently localized in the matrix of another population of morphologically distinct granules, smaller and more numerous than azurophil granules. When cytochemical detection of MPO was coupled with immunologic detection of LF, LF was observed in the population of MPO-negative granules, which were identified as specific. This was confirmed on cells that were permeabilized with saponin and stained for LF and MPO before embedding. No other neutrophil organelles displayed labeling for LF; other blood cells also were unreactive for LF. In the bone marrow, myeloblast and promyelocyte granulations were not stained and LF-containing granules appeared at the myelocyte stage. In conclusion, we confirm previous biochemical and light microscopic studies by ultrastructural demonstration of LF and MPO in two categories of granules, the specific and azurophil granules, respectively. The method described in this article avoids disruption caused by cell fractionation procedures. In the future, other intragranular proteins can be localized by a similar approach. © 1985 by Grune & Stratton, Inc.

Hematopoietic progenitor cell levels were monitored in the peripheral blood and bone marrow of 30 cancer patients receiving recombinant human granulocyte-colony stimulating-factor (rG-CSF) in a phase I/II clinical trial. The absolute number of circulating progenitor cells of granulocyte-macrophage, erythroid, and megakaryocyte lineages showed a dose-related increase up to 100-fold after four days of treatment with rG-CSF and often remained elevated two days after the cessation of therapy. The relative frequency of different types of progenitor cells in peripheral blood remained unchanged. The frequency of progenitor cells in the marrow was variable after rG-CSF treatment but in most patients was slightly decreased. The responsiveness of bone marrow progenitor cells to stimulation in vitro by rG-CSF and granulocyte-macrophage colony-stimulating factor did not change significantly during rG-CSF treatment. In patients nine days after treatment with melphalan and then rG-CSF, progenitor cell levels were very low with doses of rG-CSF at or below 10 µg/kg/d, but equaled or exceeded pretreatment values when 30 or 60 µg/kg/d of rG-CSF was given,

Thrombopoietin (TPO) or Mpl ligand is the primary physiological regulator of platelet production. This cytokine is the most potent stimulator of the proliferation and differentiation of MK progenitor and precursor cells in vitro. It also acts additively or synergistically with several cytokines on progenitor cells from various hematopoietic lineages, including the primitive stem cells. The factor is an extremely potent thrombocytopoietic agent when administrated to normal animals, and it accelerates platelet and erythropoietic recovery in several models of myelosuppression. Phase I/II clinical trials are ongoing with no detectable adverse effects. Mpl ligand does not induce platelet aggregation, but it lowers the platelet sensitivity to physiological dose of agonists. In experimental mouse models, high and chronic dose of Mpl ligand results in myelofibrosis. TPO is constantly produced by the liver and the kidney; its plasmatic clearance occurs by binding to its receptor expressed on megakaryocytes and platelets. However, the full spectrum of the biological effects of this new cytokine is not fully understood, in particular its the role in the terminal stage of platelet production. In the near future, it is likely that new insights will be obtained in the physiopathological mechanisms underlying abnormal platelet production in human. [ABSTRACT FROM AUTHOR]

Interferon α (IFNα) is used to treat JAK2V617F-driven myeloproliferative neoplasms (MPNs) but rarely clears the disease. We investigated the IFNα mechanism of action focusing on PML, an interferon target and key senescence gene whose targeting by arsenic trioxide (ATO) drives eradication of acute promyelocytic leukemia. ATO sharply potentiated IFNα-induced growth suppression of JAK2V617F patient or mouse hematopoietic progenitors, which required PML and was associated with features of senescence. In a mouse MPN model, combining ATO with IFNα enhanced and accelerated responses, eradicating MPN in most mice by targeting disease-initiating cells. These results predict potent clinical efficacy of the IFNα+ATO combination in patients and identify PML as a major effector of therapy, even in malignancies with an intact PML gene.

Activation of tyrosine kinase genes is a frequent event in human hematologic malignancies. Because gene activation could be associated with gene dysregulation, we attempted to screen for activating gene mutation based on high-level gene expression. We focused our study on the Janus kinase 2 (JAK2) gene in 90 cases of acute leukemia. This strategy led to the identification of a novel JAK2-acquired mutation in a patient with Down syndrome (DS) with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This mutation involves a 5–amino acid deletion within the JH2 pseudokinase domain (JAK2ΔIREED). Expression of JAK2ΔIREED in Ba/F3 cells induced constitutive activation of the JAK-STAT pathway and growth factor–independent cell proliferation. These results highlight the JAK2 pseudokinase domain as an oncogenic hot spot and indicate that activation of the JAK-STAT pathway may contribute to lymphoid malignancies and hematologic disorders observed in children with DS.

Introduction

Introduction:

N.M. and T.D. co-first authors, J.L.V. and Hd.T. co-last authors

Autonomous activation of JAK/STAT pathway by JAK2V617F (VF) and similar mutations in myeloproliferative neoplasms (MPN) is associated with enhanced signaling of other downstream pathways including the PI3K/mTOR. Targeting the PI3K pathway resulted in inhibition of JAK2VF cells (J Cell Mol Med 2013;17:1385), and the Torc1 inhibitor Everolimus (RAD001) showed clinical benefits in a phase I/II trial (Blood 2011;118:2069). In this study we evaluated the effects of BKM120, a pan-PI3K specific inhibitor, alone and in combination with the JAK1/JAK2 inhibitor Ruxolitinib (Ruxo) and Everolimus or PP242 (Torc1/2 inhibitor), in different MPN models and correlated efficacy with the extent of STAT5 phosphorylation inhibition.

Somatic V617F oncogenic mutation of the protein kinase JAK2 is the most prevalent genetic abnormality in the three myeloproliferative neoplasms (MPNs), namely polycythemia vera (PV, 95%), essential thrombocythemia (ET, 55%) and myelofibrosis (MF, 50%). About 30% of PV patients are homozygous for this mutation due to mitotic homologous recombination (HR). JAK2 46/1 haplotype is strongly associated with the cis-aquisition of JAK2V617Fmutation. Since, HR involves most of 46/1 haplotype, JAK2V617Fand 46/1 tagging SNPs are also reduced to homozygosity. We hypothesized that 46/1 tagging SNPs, which are in complete linkage disequilibrium with JAK2, can serve as a measure of JAK2V617Fhomozygosity. 46/1 allele burden (X%) can be used to calculate the HR (HR%) that is a measure of JAK2V617F/V617Fclones [(X%-50%)x2]. JAK2V617F/V617Ffrequency and JAK2V617Fallele burden (Y%) can be then exploited to calculate the frequency of JAK2V617F/WT[(Y%-RH%)x2] and JAK2WT[100-(JAK2V617F/WT%+ JAK2V617F/V617F)] clones. The purpose of this study was to calculate the clonal frequency of WT, JAK2V617F/WTand JAK2V617F/V617Fin progenitors compartments of PV patients.

Abstract 1757