Loss of Dnmt3a increases self-renewal and resistance to pegIFN-α in JAK2-V617F-positive myeloproliferative neoplasms.

ABSTRACT: Pegylated interferon alfa (pegIFN-α) can induce molecular remissions in patients with JAK2-V617F-positive myeloproliferative neoplasms (MPNs) by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFN-α. We investigated whether DNMT3A loss leads to alterations in JAK2-V617F LT-HSC functions conferring resistance to pegIFN-α treatment in a mouse model of MPN and in hematopoietic progenitors from patients with MPN. Long-term treatment with pegIFN-α normalized blood parameters and reduced splenomegaly and JAK2-V617F chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFN-α in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared with VF were less prone to accumulate DNA damage and exit dormancy upon pegIFN-α treatment. RNA sequencing showed that IFN-α induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ than from VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFN-α signaling. Transplantations of bone marrow from pegIFN-α-treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from patients with MPN with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F-positive colonies upon IFN-α exposure, whereas in patients with JAK2-V617F alone, the percentages of JAK2-V617F-positive colonies decreased or remained unchanged. PegIFN-α combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.

The tubulin code in platelet biogenesis.

Blood platelets are small non-nucleated cellular fragments that prevent and stop hemorrhages. They are produced in the bone marrow by megakaryocytes through megakaryopoiesis. This intricate process involves profound microtubule rearrangements culminating in the formation of a unique circular sub-membranous microtubule array, the marginal band, which supports the typical disc-shaped morphology of platelets. Mechanistically, these processes are thought to be controlled by a specific tubulin code. In this review, we summarize the current knowledge on the key isotypes, notably ß1-, α4A- and α8-tubulin, and putative post-translational modifications, involved in platelet and marginal band formation. Additionally, we provide a provisional list of microtubule-associated proteins (MAPs) involved in these processes and a survey of tubulin variants identified in patients presenting defective platelet production. A comprehensive characterization of the platelet tubulin code and the identification of essential MAPs may be expected in the near future to shed new light on a very specialized microtubule assembly process with applications in platelet diseases and transfusion.

Loss of α4A- and ß1-tubulins leads to severe platelet spherocytosis and strongly impairs hemostasis in mice.

Native circulating blood platelets present with a discoid flat morphology maintained by a submembranous peripheral ring of microtubules, named marginal band. The functional importance of this particular shape is still debated, but it was initially hypothesized to facilitate platelet interaction with the injured vessel wall and to contribute to hemostasis. The importance of the platelet discoid morphology has since been questioned on the absence of clear bleeding tendency in mice lacking the platelet-specific ß1-tubulin isotype, which exhibits platelets with a thinner marginal band and an ovoid shape. Here, we generated a mouse model inactivated for ß1-tubulin and α4A-tubulin, an α-tubulin isotype strongly enriched in platelets. These mice present with fully spherical platelets completely devoid of a marginal band. In contrast to the single knockouts, the double deletion resulted in a severe bleeding defect in a tail-clipping assay, which was not corrected by increasing the platelet count to normal values by the thrombopoietin-analog romiplostim. In vivo, thrombus formation was almost abolished in a ferric chloride-injury model, with only a thin layer of loosely packed platelets, and mice were protected against death in a model of thromboembolism. In vitro, platelets adhered less efficiently and formed smaller-sized and loosely assembled aggregates when perfused over von Willebrand factor and collagen matrices. In conclusion, this study shows that blood platelets require 2 unique α- and ß-tubulin isotypes to acquire their characteristic discoid morphology. Lack of these 2 isotypes has a deleterious effect on flow-dependent aggregate formation and stability, leading to a severe bleeding disorder.

Glycoprotein V : the unsolved GPV puzzle.

Glycoprotein V (GPV) is a highly expressed 82 KDa platelet surface transmembrane protein which is loosely attached to the GPIb-IX complex. Despite remaining questions concerning its function, GPV presents several unique features which have repercussions in hematology, atherothrombosis, immunology and transfusion. GPV is specifically expressed in platelets and megakaryocytes and is an ideal marker and reporter gene for the late stages of megakaryopoiesis. The ectodomain of GPV can be released by a number of proteases, namely thrombin, elastase and ADAM10 and 17. Although it was originally proposed as a thrombin receptor, this hypothesis was abandoned since thrombin activation was preserved after blockade of GPV cleavage and in Gp5 knockout mice. The combined potential of GPV to reflect the direct action of thrombin, platelet exposure to strong agonists and inflammatory conditions has led one to evaluate its utility as a marker in the context of atherothrombosis. Increased plasma levels of soluble GPV have notably been recorded in myocardial infarction, stroke and venous thromboembolism. It is also highly valued in transfusion to monitor platelet storage lesions. GPV presents several polymorphisms, which are a possible source of alloantibodies, while autoantibodies have been frequently detected in immune thrombocytopenia. The real biological function of this glycoprotein nevertheless remains an enigma, despite the respectively decreased and increased responses to low concentrations of collagen and thrombin observed in Gp5 knockout mice. Current studies are exploring its role in modulating general or VWF-induced platelet signaling, which could bear relevance in thrombosis and platelet clearance.

The glutaminase inhibitor CB-839 targets metabolic dependencies of JAK2-mutant hematopoiesis in MPN.

ABSTRACT: Hyperproliferation of myeloid and erythroid cells in myeloproliferative neoplasms (MPN) driven by the JAK2-V617F mutation is associated with altered metabolism. Given the central role of glutamine in anabolic and catabolic pathways, we examined the effects of pharmacologically inhibiting glutaminolysis, that is, the conversion of glutamine (Gln) to glutamate (Glu), using CB-839, a small molecular inhibitor of the enzyme glutaminase (GLS). We show that CB-839 strongly reduced the mitochondrial respiration rate of bone marrow cells from JAK2-V617F mutant (VF) mice, demonstrating a marked dependence of these cells on Gln-derived ATP production. Consistently, in vivo treatment with CB-839 normalized blood glucose levels, reduced splenomegaly and decreased erythrocytosis in VF mice. These effects were more pronounced when CB-839 was combined with the JAK1/2 inhibitor ruxolitinib or the glycolysis inhibitor 3PO, indicating possible synergies when cotargeting different metabolic and oncogenic pathways. Furthermore, we show that the inhibition of glutaminolysis with CB-839 preferentially lowered the proportion of JAK2-mutant hematopoietic stem cells (HSCs). The total number of HSCs was decreased by CB-839, primarily by reducing HSCs in the G1 phase of the cell cycle. CB-839 in combination with ruxolitinib also strongly reduced myelofibrosis at later stages of MPN. In line with the effects shown in mice, proliferation of CD34+ hematopoietic stem and progenitor cells from polycythemia vera patients was inhibited by CB-839 at nanomolar concentrations. These data suggest that inhibiting GLS alone or in combination with inhibitors of glycolysis or JAK2 inhibitors represents an attractive new therapeutic approach to MPN.

Platelets and neutrophils cooperate to induce increased neutrophil extracellular trap formation in JAK2V617F myeloproliferative neoplasms.

BACKGROUND: Neutrophils participate in the pathogenesis of thrombosis through the formation of neutrophil extracellular traps (NETs). Thrombosis is the main cause of morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). Recent studies have shown an increase in NET formation (NETosis) both in patients with JAK2V617F neutrophils and in mouse models, and reported the participation of NETosis in the pathophysiology of thrombosis in mice. OBJECTIVES: This study investigated whether JAK2V617F neutrophils are sufficient to promote thrombosis or whether their cooperation with other blood cell types is necessary. METHODS: NETosis was studied in PF4iCre;Jak2V617F/WT mice expressing JAK2V617F in all hematopoietic lineages, as occurs in MPNs, and in MRP8Cre;Jak2V617F/WT mice in which JAK2V617F is expressed only in leukocytes. RESULTS: In PF4iCre;Jak2V617F/WT mice, an increase in NETosis and spontaneous lung thrombosis abrogated by DNAse administration were observed. The absence of spontaneous NETosis or lung thrombosis in MRP8Cre;Jak2V617F/WT mice suggested that mutated neutrophils alone are not sufficient to induce thrombosis. Ex vivo experiments demonstrated that JAK2V617F-mutated platelets trigger NETosis by JAK2V617F-mutated neutrophils. Aspirin treatment in PF4iCre;Jak2V617F/WT mice reduced NETosis and reduced lung thrombosis. In cytoreductive-therapy-free patients with MPN treated with aspirin, plasma NET marker concentrations were lower than that in patients with MPN not treated with aspirin. CONCLUSION: Our study demonstrates that JAK2V617F neutrophils alone are not sufficient to promote thrombosis; rather, platelets cooperate with neutrophils to promote NETosis in vivo. A new role for aspirin in thrombosis prevention in MPNs was also identified.

IL-1ß promotes MPN disease initiation by favoring early clonal expansion of JAK2-mutant hematopoietic stem cells.

ABSTRACT: JAK 2-V617F is the most frequent somatic mutation causing myeloproliferative neoplasm (MPN). JAK2-V617F can be found in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) with a frequency much higher than the prevalence of MPNs. The factors controlling the conversion of JAK2-V617F CHIP to MPN are largely unknown. We hypothesized that interleukin-1ß (IL-1ß)-mediated inflammation can favor this progression. We established an experimental system using bone marrow (BM) transplantations from JAK2-V617F and GFP transgenic (VF;GFP) mice that were further crossed with IL-1ß-/- or IL-1R1-/- mice. To study the role of IL-1ß and its receptor on monoclonal evolution of MPN, we performed competitive BM transplantations at high dilutions with only 1 to 3 hematopoietic stem cells (HSCs) per recipient. Loss of IL-1ß in JAK2-mutant HSCs reduced engraftment, restricted clonal expansion, lowered the total numbers of functional HSCs, and decreased the rate of conversion to MPN. Loss of IL-1R1 in the recipients also lowered the conversion to MPN but did not reduce the frequency of engraftment of JAK2-mutant HSCs. Wild-type (WT) recipients transplanted with VF;GFP BM that developed MPNs had elevated IL-1ß levels and reduced frequencies of mesenchymal stromal cells (MSCs). Interestingly, frequencies of MSCs were also reduced in recipients that did not develop MPNs, had only marginally elevated IL-1ß levels, and displayed low GFP-chimerism resembling CHIP. Anti-IL-1ß antibody preserved high frequencies of MSCs in VF;GFP recipients and reduced the rate of engraftment and the conversion to MPN. Our results identify IL-1ß as a potential therapeutic target for preventing the transition from JAK2-V617F CHIP to MPNs.

Mutations in the most divergent α-tubulin isotype, α8-tubulin, cause defective platelet biogenesis.

BACKGROUND: In the panel of genes commonly associated with inherited macrothrombocytopenia, an important fraction encodes key cytoskeletal proteins such as tubulin isotypes, the building blocks of microtubules. Macrothrombocytopenia-causing mutations have been identified in the TUBB1 and TUBA4A genes, emphasizing their importance in the formation of platelets and their marginal band, a unique microtubule ring-like structure that supports the platelet typical disc-shaped morphology. This raised the hypothesis that other tubulin isotypes normally expressed in platelets could play a similar role in their formation. OBJECTIVES: To assess whether tubulin isotype genes other than TUBA4A and TUBB1 could be implicated in inherited macrothrombocytopenia. METHODS: We used high throughput sequencing to screen a cohort of 448 French blood donors with mild thrombocytopenia for mutations in a panel of selected genes known or suspected to be involved in platelet biogenesis. RESULTS: We identified six distinct novel mutations in TUBA8, which encodes the most-divergent α-tubulin, as the causative determinant of macrothrombocytopenia and platelet marginal band defects. Functionally, all TUBA8 mutations were found to fully or partially inhibit the incorporation of the mutated α8-tubulin in the microtubule network. CONCLUSION: This study provides strong support for a key role of multiple tubulin genes in platelet biogenesis by discovering variants in a tubulin gene that was previously not known to be important for platelets.

Isolation of Mouse Megakaryocyte Progenitors.

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

An essential role for α4A-tubulin in platelet biogenesis.

During platelet biogenesis, microtubules (MTs) are arranged into submembranous structures (the marginal band) that encircle the cell in a single plane. This unique MT array has no equivalent in any other mammalian cell, and the mechanisms responsible for this particular mode of assembly are not fully understood. One possibility is that platelet MTs are composed of a particular set of tubulin isotypes that carry specific posttranslational modifications. Although ß1-tubulin is known to be essential, no equivalent roles of α-tubulin isotypes in platelet formation or function have so far been reported. Here, we identify α4A-tubulin as a predominant α-tubulin isotype in platelets. Similar to ß1-tubulin, α4A-tubulin expression is up-regulated during the late stages of megakaryocyte differentiation. Missense mutations in the α4A-tubulin gene cause macrothrombocytopenia in mice and humans. Defects in α4A-tubulin lead to changes in tubulin tyrosination status of the platelet tubulin pool. Ultrastructural defects include reduced numbers and misarranged MT coils in the platelet marginal band. We further observed defects in megakaryocyte maturation and proplatelet formation in Tuba4a-mutant mice. We have, thus, discovered an α-tubulin isotype with specific and essential roles in platelet biogenesis.