The JAK2V617F mutation disrupts the regulated association between calreticulin and the glucocorticoid receptor observed in normal erythroid cells

Calreticulin (CALR) is a multifunctional protein normally found within the lumen of the endoplasmic reticulum that mediates the cellular response to Ca2+ by chaperoning other proteins to their acting sites. Somatic loss-of-function mutations in the CALR gene were recently discovered in 70% of patients with the Philadelphia-negative myeloproliferative neoplasm (MPN) primary myelofibrosis (PMF) who did not harbor gain-of-function mutations of JAK21,2. Nevertheless, the JAK2 pathway is constitutively activated also in patients carrying CALR mutation and treatments with JAK2 inhibitors are effective not only in MPN patients (PMF and polycythemia vera, PV) harboring JAK2 mutations but also in PMF patients harboring mutations in CALR3. We have previously reported that erythroid cells from PV and PMF patients express abnormal activity of the glucocorticoid receptor (GR), a nuclear receptor whose transcriptional activity plays an important role in the regulation of stress erythropoiesis4,5. Since GR is one of the numerous proteins regulated by CALR6, we hypothesized that in human erythroid cells CALR regulates GR functions and that this regulation is disrupted both by CALR and JAK2 mutations in MPN. In this study we tested this hypothesis by determining whether GR and CALR are associated in normal erythroid cells and whether this association is impaired in those from MPN patients. First, biochemical studies determined that human erythroblasts (Erys) expanded ex-vivo from normal stem cell sources [cord blood (CB) and adult blood (AB)] and from MPN patients contain similar levels of CALR and GR. Analyses of cell fractions indicated that in normal Erys, CALR was constitutively localized in the cytoplasm while GR was detected either in the cytoplasm or in the nucleus, depending on the growth factor (the glucocorticoid receptor agonist dexamethasone, erythropoietin or stem cell factor) to which they had been exposed. Second, robust levels of CALR and GR expression were also detected by confocal microscopy. In addition, this analyses revealed that in Erys expanded from normal sources CALR and GR are co-localized in the cytoplasm and that the cytoplasmic association between the two proteins is increased by growth factor deprivation and further enhanced by stimulation with growth factors that activate the JAK2/STAT5 signaling (dexamethasone and/or erythropoietin) while it is inhibited by stimulation with factors that do not use this pathway (stem cell factor). By contrast, in Erys expanded from MPN carrying either CALR or JAK2 mutations, CALR and GR are not associated and remain not associated when the cells are exposed to dexamethasone or erythropoietin. However, in Erys from JAK2V17F-positive MPN patients, association between CALR and GR in the cytoplasm is restored by exposing the cells to the JAK2 inhibitor ruxolitinib. These results suggest that CALR/GR association is a downstream event induced by the JAK2/STAT5 pathway and identify for the first time that CALR functions are impaired in erythroid cells from MPN patients carrying JAK2 mutations

Whirling Platelets Away for Transfusion

With a growing demand for platelet transfusions, large-scale ex vivo platelet production would alleviate the reliance on donors. Now, Ito et al. report that turbulence is an important physical regulator of platelet generation in vivo and can be exploited in a bioreactor to enable clinical scale production of functional platelets starting from human iPSCs. With a growing demand for platelet transfusions, large-scale ex vivo platelet production would alleviate the reliance on donors. Now, Ito et al. report that turbulence is an important physical regulator of platelet generation in vivo and can be exploited in a bioreactor to enable clinical scale production of functional platelets starting from human iPSCs

Stathmin 1 deficiency induces erythro-megakaryocytic defects leading to macrocytic anemia and thrombocythemia in Stathmin 1 knock out mice

Stathmin 1 (STMN1) is a cytosolic phosphoprotein that was discovered as a result of its high level of expression in leukemic cells. It plays an important role in the regulation of mitosis by promoting depolymerization of the microtubules that make up the mitotic spindle and, aging has been shown to impair STMN1 levels and change microtubule stability. We have previously demonstrated that a high level of STMN1 expression during early megakaryopoiesis is necessary for proliferation of megakaryocyte progenitors and that down-regulation of STMN1 expression during late megakaryopoiesis is important for megakaryocyte maturation and platelet production. In this report, we examined the effects of STMN1 deficiency on erythroid and megakaryocytic lineages in the mouse. Our studies show that STMN1 deficiency results in mild thrombocytopenia in young animals which converts into profound thrombocythemia as the mice age. STMN1 deficiency also lead to macrocytic changes in both erythrocytes and megakaryocytes that persisted throughout the life of STMN1 knock-out mice. Furthermore, STMN1 knock-out mice displayed a lower number of erythroid and megakaryocytic progenitor cells and had delayed recovery of their blood counts after chemotherapy. These studies show an important role for STMN1 in normal erythro-megakaryopoietic development and suggests potential implications for disorders affecting these hematopoietic lineages

The role of stathmin, a regulator of mitosis, in hematopoiesis

Steel, JC ORCiD: 0000-0003-3608-7542Introduction Stathmin is a 17KDa cytosolic protein that plays an important role in the regulation of microtubule dynamics, mitotic spindle formation, cell cycle progression and cell differentiation. Stathmin knockout (KO) mice were initially reported to have a normal phenotype but were subsequently shown to develop an age-related neurological phenotype with axonopathy evident in both central and peripheral nervous systems. These mice were also shown to have a defect in recovery from acute ischemic renal injury. We had previously shown that stathmin plays an important role in the differentiation and proliferation of megakaryocytes (MK) and that down-regulation of stathmin is necessary for the maturation of MK and platelet production in vitro. In this study, we investigated the role of stathmin in megakaryopoiesis and hematopoiesis in vivo using the stathmin KO mouse as an experimental model. Results Stathmin KO mice had lower platelet (PLT) counts at 3 weeks of age when compared to WT mice. The WT mice had a mean PLT count of 662 ± 27 K/μL while KO mice had a mean PLT count of 543 ± 37 K/μL. This correlated with larger and fewer MK in the bone marrow of KO mice (WT: 4.2 ± 0.7 MK/40X field; KO: 3.6 ± 0.2 MK/40X field). Furthermore, in the spleen, there was a 10 fold decrease in the number of MK in KO mice compared to WT mice (6.6 ± 0.6 vs 0.7 ± 0.1 MK/40X field). By 8 weeks, PLT counts and MK size and numbers in the bone marrow and spleen were similar in WT and KO mice. Interestingly, by 16 weeks, the mean PLT of KO mice became significantly higher than that of WT and by 40 weeks, the mean PLT count of KO mice was 1379 ± 100K/μL compared to 1045 ± 120K/μL in WT mice (P<0.05). Microscopic analysis of the bone marrow at 46 weeks of age showed approximately 50% more MK in KO mice compared to WT mice. Differences in red blood cell counts (RBC) were also observed. While at 3 weeks, there were no significant differences between the 2 groups, at 8 weeks, KO mice had significantly lower RBC counts, hemoglobin levels (Hb) and hematocrit (HCT). This trend continued until the last measurement recorded at 40 weeks. Mean RBC in WT mice was 10.5 ± 0.1M/μL compared to 8.9 ± 0.2M/μL in KO mice. The mean corpuscular volume (MCV) and the red blood cell distribution width (RDW) were consistently higher in KO mice than in WT mice. No significant differences were noted in white blood cell counts. Bone marrow cell counts were significantly lower in KO mice when compared to WT mice at different ages from 3–40 weeks. Progenitor cell assays from 10–12 week old animals have shown that bone marrow from KO mice produce significantly fewer BFU-E and Pre-B colonies while no differences were observed in CFU-GMs. Conclusions The phenotypic characteristics of stathmin KO mice confirmed our prior in vitro findings that suggested a role for stathmin in megakaryopoiesis. We expected to see a decrease in the number of platelets and MK coupled with an increase in MK size. This was confirmed in stathmin KO mice at 3 weeks of age. However, we did not expect to see the marked increase in the number of platelets and MK that was observed as the mice aged. The exact mechanism for this has not been identified. Interestingly, the stathmin KO mice exhibited characteristic features of megaloblastic anemia including mild anemia and a significant increase in MCV and RDW. The megaloblastic anemia that is seen in the presence of B12 and folate deficiency results from interference with DNA synthesis resulting in asynchronous maturation of the nucleus and the cytoplasm. We believe a similar phenomenon is occurring in the stathmin KO mice. The deficiency of stathmin results in aberrant exit from mitosis, thereby delaying nuclear maturation and resulting in the megaloblastic features. Thus, the deficiency of stathmin in the KO mice results in two hematopoietic phenotypes that are seen in humans, megaloblastic anemia and thrombocytosis. It is unclear whether mutations of stathmin in humans might result in similar phenotypes. This is a question that will require further investigation. Future studies will investigate the compensatory mechanisms that result in the switch from decreased to increased platelet production as the mice age. Furthermore, examining the effects of hematopoietic stress (e.g. response to chemotherapy or bleeding) in stathmin KO mice might also elucidate a role for stathmin in the recovery from hematopoietic injury as was seen in acute ischemic renal injury

Down-regulation of stathmin expression is required for megakaryocyte maturation and platelet production

The final stages of of megakaryocyte (MK) maturation involve a series of steps, including polyploidization and proplatelet formation. Although these processes are highly dependent on dynamic changes in the microtubule (MT) cytoskeleton, the mechanisms responsible for regulation of MTs in MKs remain poorly defined. Stathmin is a highly conserved MT-regulatory protein that has been suggested to play a role in MK differentiation of human leukemic cell lines. However, previous studies defining this relationship have reached contradictory conclusions. In this study, we addressed this controversy and investigated the role of stathmin in primary human MKs. To explore the importance of stathmin down-regulation during megakaryocytopoiesis, we used a lentiviral-mediated gene delivery system to prevent physiologic down-regulation of stathmin in primary MKs. We demonstrated that sustained expression of constitutively active stathmin delayed cytoplasmic maturation (ie, glycoprotein GPIb and platelet factor 4 expression) and reduced the ability of MKs to achieve high levels of ploidy. Moreover, platelet production was impaired in MKs in which down-regulation of stathmin expression was prevented. These studies indicate that suppression of stathmin is biologically important for MK maturation and platelet production and support the importance of MT regulation during the final stages of thrombopoiesis

Harnessing a Novel Dyrk1a-Ablim2-MKL1 Regulatory Module in Megakaryocyte Morphogenesis to Enable Scalable Platelet "Pharming"

International audienceGrowing clinical demands for platelet transfusions combined with supply limitations have created shortages which are trending toward a global crisis. Major efforts have been taken to address key issues of platelet sources, storage, and utilization. Recent progress in ex vivo culture-based production of megakaryocytes (Mk) and platelets, "pharming," has highlighted the potential for novel, donor-independent sources amenable to antigenic editing and cryo-stockpiling. Such cultures can be easily initiated from umbilical cord blood (CB) progenitors, induced pluripotent stem cells (iPSC), or directly re-programmed somatic cells. The major roadblock associated with these Mk sources consists of their fetal ontogenic status, which is beneficial for expansion but severely limits platelet production. The ability to elicit in pre-expanded Mk an adult program of morphogenesis (polyploidization, enlargement, and proplatelet formation) would enable circumvention of this scalability barrier. A master regulator of adult Mk morphogenesis consists of the transcriptional coactivator MKL1 which undergoes nuclear translocation in response to RhoA-mediated actin polymerization, stimulated by thrombopoietin and environmental mechano-sensing. Nuclear MKL1 associates with the transcription factor SRF1 to upregulate cytoskeletal remodeling factors, including filamin A and Hic-5, that act as morphogenesis effectors. Our previous studies identified in infantile CB Mk a failure in MKL1 upregulation resulting from repression by the oncofetal RNA-binding factor IGF2BP3. Pharmacologic suppression of IGF2BP3 with BET inhibitors rescued MKL1 expression and improved platelet production but caused cycle arrest preventing polyploidization. As an alternative approach to abrogate the fetal blockade in Mk morphogenesis, we sought to promote MKL1 activity by targeting a kinase, Dyrk1a, which had been shown to restrain MKL1 from nuclear translocation. Treatment of infantile CB Mk with a variety of Dyrk1-selective inhibitors including harmine and EHT 1610 strongly enhanced polyploidization (p = 0.015 and 0.009 respectively), enlargement (p < 0.005) , and in vitro platelet release (2 fold each, p = 0.001 and 0.007 respectively), attaining levels seen with adult Mk. When xenotransplanted into NSG mice, harmine-treated CB Mk demonstrated enhanced capability for in vivo platelet release (about 5 fold, p = 0.016). CB stem cells expanded with the AHR antagonist SR1 and an iPSC-Mk cell line also responded to Dyrk1 inhibition with robustly increased morphogenesis. Several findings implicated MKL1 in this response: 1) induction of nuclear translocation by the inhibitors, 2) induction of target genes (filamin A and Hic-5) by the inhibitors, and 3) loss of response to inhibitors in Mkl1-ko murine progenitors. Supporting Dyrk1a as a relevant target, mice with Mk-specific loss of one Dyrk1a allele (Dyrk1aflox/wt;Pf4-Cre) displayed increases in platelet counts (p = 0.037) and marrow Mk polyploidization (p = 0.02). In addition, retroviral expression in human progenitors of a dominant negative Dyrk1a mutant K188R promoted Mk enlargement (p = 0.014). shRNA knockdowns could not be obtained due to toxicity of > ~60% loss of Dyrk1a. To determine mechanisms for Dyrk1a control of morphogenesis, we analyzed the actin cytoskeleton, a key regulator of MKL1. Dyrk1 inhibition in all types of Mk progenitors (adult, infantile, and iPSC) induced assembly of cortical filamentous actin (F-actin), as detected by Alexa594-phalloidin staining. Prior studies showed cytoskeletal binding by Dyrk1a and direct phosphorylation of F-actin regulators N-WASP and Ablim1. A survey of human marrow expression patterns for candidate Dyrk1a substrates (The Human Protein Atlas) identified Ablim2, as showing a Mk-specific, cortical staining pattern. Dyrk1 inhibition increased Ablim2 levels ~5-fold in CB Mk (p < 0.005), and immunofluorescence displayed a cortical distribution similar to F-actin. Lentiviral shRNA knockdown of Ablim2 abrogated all effects of Dyrk1 inhibition, blocking: F-actin formation, MKL1 nuclear translocation, activation of the MKL1 targets, and Mk morphogenesis. These findings thus delineate a novel Dyrk1a-Ablim2-MKL1 regulatory module in Mk morphogenesis that can be manipulated to address the problem of scaling ex vivo production and might also serve as a future in vivo therapeutic target for thrombocytopenia

Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition

Hematopoietic transitions that accompany fetal development, such as erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors do not execute the adult morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects may also contribute to inferior platelet recovery after cord blood stem cell transplantation and may underlie inefficient platelet production by megakaryocytes derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors has identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism that is known to drive adult megakaryocyte morphogenesis. This blockade resulted from neonatal-specific expression of an oncofetal RNA-binding protein, IGF2BP3, which prevented the destabilization of the nuclear RNA 7SK, a process normally associated with adult megakaryocytic P-TEFb activation. Knockdown of IGF2BP3 sufficed to confer both phenotypic and molecular features of adult-type cells on neonatal megakaryocytes. Pharmacologic inhibition of IGF2BP3 expression via bromodomain and extraterminal domain (BET) inhibition also elicited adult features in neonatal megakaryocytes. These results identify IGF2BP3 as a human ontogenic master switch that restricts megakaryocyte development by modulating a lineage-specific P-TEFb activation mechanism, revealing potential strategies toward enhancing platelet production.NIH [DK090926, HL130550, T32 CA009109-39]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]