Amyloid precursor protein 770 is specifically expressed and released from platelets.

Platelets not only play an essential role in hemostasis after vascular injury but are also involved in the development of coronary artery disease (CAD) and cerebrovascular lesions. Patients with CAD and cerebral ischemia are recommended to undergo antiplatelet therapy, but they have an increased incidence of major bleeding complications. Both assessment of the platelet activation status and response to antiplatelet therapy in each patient are highly desired. ß-Amyloid precursor protein (APP) 770 is expressed in vascular endothelial cells, and its extracellular region, a soluble form of APP770 (sAPP770, also called nexin-2), is proteolytically cleaved for shedding. Abundant sAPP770 is also released from activated platelets. In this study, we used peripheral blood samples from patients with CAD and control subjects and evaluated sAPP770 as a specific biomarker for platelet activation. First, the plasma levels of sAPP770 correlated well with those of the soluble form CD40 ligand (CD40L), an established biomarker for platelet activation. Additionally, flow cytometry analysis using peripheral blood cells showed that CD40L expression is up-regulated in activated T cells, whereas APP770 expression is negligible in all blood cell types except platelets. Following stimulation with collagen or ADP, aggregating platelets immediately released sAPP770. Finally, patients with dual antiplatelet therapy showed significantly lower levels of plasma sAPP770 than those with no therapy. Taken together, our data show that plasma sAPP770 could be a promising biomarker for platelet activation.

Optimal timing of apheresis for the efficient mobilization of peripheral blood progenitor cells recruited by high-dose granulocyte colony-stimulating factor in healthy donors.

Predictors of peripheral blood stem cell (PBSC) yield can potentially improve the comfort, safety, and efficacy of CD34+ cell collection from donors treated with recombinant human granulocyte colony-stimulating factor (G-CSF). We investigated 181 apheresis procedures on 109 healthy allogeneic donors to identify factors correlating with efficient PBSC collection. Apheresis started on Day 4 or 5 and continued up to Day 6 of G-CSF administration. CD34+ cell　yields on Days 4 and 5 were comparable, and significantly higher than on Day 6. This suggests that starting apheresis on Day 4 rather than Day 5 may be preferable, to reduce G-CSF exposure and optimize yield, even if multi-day collection is required. More CD34+ cells were collected from male and cytomegalovirus (CMV)-seronegative donors than from female and CMV-seropositive donors, respectively. The yields of CD34+ cells were similarly high in both male and female donors aged 20-29 years; yields decreased in female donors in their thirties, and were comparably low in both male and female donors in their forties and thereafter. These findings should guide decision-making about when to begin apheresis, and encourage careful consideration of donor factors such as gender, age, and CMV serostatus when collecting PBSCs.

Mechanisms of Impaired Neutrophil Migration by MicroRNAs in Myelodysplastic Syndromes.

In myelodysplastic syndromes (MDS), functional defects of neutrophils result in high mortality because of infections; however, the molecular basis remains unclear. We recently found that miR-34a and miR-155 were significantly increased in MDS neutrophils. To clarify the effects of the aberrant microRNA expression on neutrophil functions, we introduced miR-34a, miR-155, or control microRNA into neutrophil-like differentiated HL60 cells. Ectopically introduced miR-34a and miR-155 significantly attenuated migration toward chemoattractants fMLF and IL-8, but enhanced degranulation. To clarify the mechanisms for inhibition of migration, we studied the effects of miR-34a and miR-155 on the migration-regulating Rho family members, Cdc42 and Rac1. The introduced miR-34a and miR-155 decreased the fMLF-induced active form of Cdc42 to 29.0 ± 15.9 and 39.7 ± 4.8% of that in the control cells, respectively, although Cdc42 protein levels were not altered. miR-34a decreased a Cdc42-specific guanine nucleotide exchange factor (GEF), dedicator of cytokinesis (DOCK) 8, whereas miR-155 reduced another Cdc42-specific GEF, FYVE, RhoGEF, and PH domain-containing (FGD) 4. The knockdown of DOCK8 and FGD4 by small interfering RNA suppressed Cdc42 activation and fMLF/IL-8-induced migration. miR-155, but not miR-34a, decreased Rac1 protein, and introduction of Rac1 small interfering RNA attenuated Rac1 activation and migration. Neutrophils from patients showed significant attenuation in migration compared with healthy cells, and protein levels of DOCK8, FGD4, and Rac1 were well correlated with migration toward fMLF (r = 0.642, 0.686, and 0.436, respectively) and IL-8 (r = 0.778, 0.659, and 0.606, respectively). Our results indicated that reduction of DOCK8, FGD4, and Rac1 contributes to impaired neutrophil migration in MDS.

Autocrine and Paracrine Interactions between Multiple Myeloma Cells and Bone Marrow Stromal Cells by Growth Arrest-specific Gene 6 Cross-talk with Interleukin-6.

The pathogenesis of multiple myeloma (MM) has not yet been fully elucidated. Our microarray analysis and immunohistochemistry revealed significant up-regulation of growth arrest-specific gene 6 (Gas6), a vitamin K-dependent protein with a structural homology with protein S, in bone marrow (BM) cells of MM patients. ELISA showed that the serum levels of soluble Gas6 were significantly increased in the MM patients when compared with healthy controls. Gas6 was overexpressed in the human CD138-positive MM cell line RPMI-8226. Exogenous Gas6 suppressed apoptosis induced by serum deprivation and enhanced cell proliferation of the MM cells. The conditional medium from the human BM stromal cell line HS-5 induced cell proliferation and anti-apoptosis of the MM cells with extracellular signal-regulated kinase, Akt, and nuclear factor-κB phosphorylation, which were reversed by the neutralizing antibody to Gas6 or IL-6. The TAM family receptor Mer, which has been identified as a Gas6 receptor, was overexpressed in BM cells of MM patients. The knockdown of Mer by siRNA inhibited cell proliferation, anti-apoptosis, and up-regulation of intercellular cell adhesion molecule-1 (ICAM-1) in MM cells stimulated by an HS-5 cell-conditioned medium. Furthermore, the Gas6-neutralizing antibody reduced the up-regulation of IL-6 and ICAM-1 induced by a HS-5 cell-conditioned medium in MM cells. The present study provides new evidence that autocrine and paracrine stimulation of Gas6 in concert with IL-6 contributes to the pathogenesis of MM, suggesting that Gas6-Mer-related signaling pathways may be a promising novel target for treating MM.

Pretreatment evaluation of fluorescence resonance energy transfer-based drug sensitivity test for patients with chronic myelogenous leukemia treated with dasatinib.

Tyrosine kinase inhibitors (TKI) are used for primary therapy in patients with newly diagnosed CML. However, a reliable method for optimal selection of a TKI from the viewpoint of drug sensitivity of CML cells has not been established. We have developed a FRET-based drug sensitivity test in which a CrkL-derived fluorescent biosensor efficiently quantifies the kinase activity of BCR-ABL of living cells and sensitively evaluates the inhibitory activity of a TKI against BCR-ABL. Here, we validated the utility of the FRET-based drug sensitivity test carried out at diagnosis for predicting the molecular efficacy. Sixty-two patients with newly diagnosed chronic phase CML were enrolled in this study and treated with dasatinib. Bone marrow cells at diagnosis were subjected to FRET analysis. The ΔFRET value was calculated by subtraction of FRET efficiency in the presence of dasatinib from that in the absence of dasatinib. Treatment response was evaluated every 3 months by the BCR-ABL1 International Scale. Based on the ΔFRET value and molecular response, a threshold of the ΔFRET value in the top 10% of FRET efficiency was set to 0.31. Patients with ΔFRET value ≥0.31 had significantly superior molecular responses (MMR at 6 and 9 months and both MR4 and MR4.5 at 6, 9, and 12 months) compared with the responses in patients with ΔFRET value <0.31. These results suggest that the FRET-based drug sensitivity test at diagnosis can predict early and deep molecular responses. This study is registered with UMIN Clinical Trials Registry (UMIN000006358).

Efficacy of D- red blood cell transfusion and rituximab therapy in autoimmune hemolytic anemia with anti-D and panreactive autoantibodies arising after hematopoietic stem cell transplant.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) is caused by autoantibodies to red blood cells (RBCs), which can be panreactive and/or specific to Rh/other blood group antigens. We report a severe case of AIHA after bone marrow transplantation (BMT) due to autoanti-D triggered by reactivation of Epstein-Barr virus (EBV) infection. A combined strategy of D- RBC transfusion and administration of anti-CD20 monoclonal antibody (MoAb) resolved the hemolysis. CASE REPORT: A 33-year-old male underwent allogeneic BMT from an ABO-identical and HLA-matched unrelated male donor. Five months later, while having mild chronic graft-versus-host disease, he manifested AIHA, with a hemoglobin (Hb) level of 5.1 g/dL on AIHA Day 2 (Posttransplant Day 156) and was refractory to D+ RBCs, with a Hb level of 2.4 g/dL on AIHA Day 6. Anti-D-like autoantibodies (titer 1280, subclass immunoglobulin G1 , monocyte monolayer assay 28.7%) and panreactive (titer 40) were identified. Changing the RBC transfusion strategy to D- increased his Hb level to 6.7 g/dL on Day 10. Administration of anti-CD20 MoAb mitigated EBV-related B-cell proliferation and reduced anti-D autoantibody titer to 320 by Day 16 with normalized Hb concentration after 6 months. CONCLUSION: In severe AIHA, when standard treatment and regular RBC transfusions are ineffective, transfusion of RBCs lacking the target antigen(s) of autoantibodies and administration of anti-CD20 MoAb should be considered.

[Airway obstruction due to localized tracheal lesion of extranodal NK/T-cell lymphoma, nasal type].

A 76-year-old man presented with a tracheal tumor associated with severe respiratory obstruction. A tracheotomy was performed due to respiratory failure. F-fluorodeoxyglucose (FDG) -positron emission tomography/computed tomography revealed an abnormal accumulation of FDG (maximum standardized uptake value: 16) in the trachea. A histopathological examination of the tracheal biopsy revealed extranodal NK/T-cell lymphoma, nasal type (ENKL). He was treated with concurrent radiotherapy (50 Gy) for the tracheal tumor and three courses of two-thirds dose ofdexamethasone, etoposide, ifosfamide, and carboplatin. Although the tumor responded remarkably well to this therapy, the patient died of an ENKL recurrence in the lungs and liver 11 months post therapy.

A possible role of low regulatory T cells in anti-acetylcholine receptor antibody positive myasthenia gravis after bone marrow transplantation.

BACKGROUND: Chronic graft-versus-host disease (GVHD) appears several months following allogenic hematopoietic stem cell transplantation (HSCT) and is clinically analogous to autoimmune disorder. Polymyositis is a common neuromuscular disorder in chronic GVHD, but myasthenia gravis (MG) is extremely rare. Hence, its pathophysiology and treatment have not been elucidated. CASE PRESENTATION: A 63-year-old man with a history of chronic GVHD presented with ptosis, dropped head, and dyspnea on exertion, which had worsened over the previous several months. He showed progressive decrement of compound muscle action potential in the deltoid muscle evoked by 3-Hz repetitive nerve stimulation, a positive edrophonium test, and elevated levels of serum anti-acetylcholine receptor antibodies, which suggested a diagnosis of generalized MG. No thymoma was found. Flow cytometric analysis revealed a remarkable depletion of peripheral Tregs (CD4+CD25highFOXP3+ cells, 0.24% of the total lymphocytes). Administration of prednisolone and tacrolimus was insufficient to alleviate his symptoms; however, the use of rituximab successfully improved his condition. CONCLUSIONS: Myasthenic symptoms appeared in the process of tapering prednisolone for the treatment of chronic GVHD, supporting the diagnosis of MG associated with chronic GVHD. The present case proposes a possibility that reduction of Tregs might contribute to the pathogenesis of MG underlying chronic GVHD. Immunotherapy with rituximab is beneficial for treatment of refractory MG and GVHD.

Pharmacokinetics and pharmacodynamics of dasatinib in the chronic phase of newly diagnosed chronic myeloid leukemia.

PURPOSE: Dasatinib is a novel, oral, multi-targeted kinase inhibitor of breakpoint cluster region-abelson (BCR-ABL) and Src family kinases. The study investigated pharmacokinetic (PK) and pharmacodynamic (PD) analyses of dasatinib in 51 newly diagnosed, chronic phase, chronic myeloid leukemia patients. METHODS: The dasatinib concentration required to inhibit 50 % of the CrkL (CT10 regulator of kinase like) phosphorylation in bone marrow CD34+ cells (half maximal (50 %) inhibitory concentration (IC50)CD34+cells) was calculated from each patient's dose-response curve using flow cytometry. PK parameters were obtained from the population pharmacokinetic analysis of dasatinib concentrations in plasma on day 28 after administration. RESULTS: Early molecular responses were not significantly associated with PK or PD (IC50 CD34+cells) parameters. However, the PK/PD parameter-time above IC50 CD34+cells-significantly correlated with BCR-ABL transcript level at 3 months (correlation coefficient (CC) = -0.292, P = 0.0375) and the reduction of BCR-ABL level at 1 or 3 months (CC = -0.404, P = 0.00328 and CC = -0.356, P = 0.0104, respectively). Patients with more than 12.6 h at time above IC50 CD34+cells achieved a molecular response of 3.0 log reduction at 3 months and those more than 12.8 h achieved a deep molecular response less than 4.0 log reduction at 6 months at a significantly high rate (P = 0.013, odds ratio = 4.8 and P = 0.024, odds ratio = 4.3, respectively). CONCLUSION: These results suggest that the anti-leukemic activity of dasatinib exhibits in a time-dependent manner and that exposure for more than 12.8 h at time above IC50 CD34+cells could significantly improve prognosis.

Dysregulation of the MIRLET7/HMGA2 axis with methylation of the CDKN2A promoter in myeloproliferative neoplasms.

Overexpression of high mobility group AT-hook 2 (Hmga2), which is negatively regulated by MIRLET7 micro RNAs through 3'-untranslated region (3'UTR), causes proliferative haematopoiesis mimicking myeloproliferative neoplasms (MPNs) and contributes to progression of myelofibrosis in mice. Thus, we investigated HMGA2 mRNA expression in 66 patients with MPNs including 23 polycythaemia vera (PV), 33 essential thrombocythaemia (ET) and 10 primary myelofibrosis (PMF). HMGA2 mRNA expression, especially variant 1 with 3'UTR that contains MIRLET7-specific sites, rather than variant 2 lacking 3'UTR, is frequently deregulated due to decreased MIRLET7 expression in granulocytes from over 20% of PV and ET, and in either granulocytes or CD34(+) cells from 100% of PMF. Patients with deregulated HMGA2 mRNA expression were significantly more likely to show splenomegaly, high serum lactate dehydrogenase values, and methylation of the CDKN2A promoter compared with other patients without deregulation of HMGA2. A histone deacetylase inhibitor, panobinostat, significantly increased MIRLET7 expression and reduced variant 1 of HMGA2 mRNA expression, but not variant 2, in both U937 cells and PMF-derived CD34(+) cells. Moreover, both panobinostat and small interfering RNA of HMGA2 demethylated the CDKN2A promoter in U937 cells. In conclusion, the frequently dysregulated MIRLET7/HMGA2 axis could be a therapeutic target in MPNs.

A prospective analysis of clinical efficacy and safety in chronic myeloid leukemia-chronic phase patients with imatinib resistance or intolerance as evaluated using European LeukemiaNet 2013 criteria.

BACKGROUND: We conducted a phase II study to evaluate the efficacy and safety of dasatinib in Japanese patients with imatinib-resistant or imatinib-intolerant chronic myeloid leukemia (CML). METHODS: From 2009 to 2011, 54 CML-chronic phase (CP) patients with resistance (n = 40) or intolerance (n = 25) to imatinib were registered to undergo dasatinib treatment. Eleven patients showed both resistance and intolerance to imatinib. Coincidentally, the resistance criteria in this study were the same as a non-optimal response to tyrosine kinase inhibitors (TKIs) as defined in the European LeukemiaNet (ELN) 2013 recommendations. RESULTS: The overall incidence rate of major molecular response (MMR) at 12 months was 62.3% (n = 47). Forty patients with resistance to imatinib who were 'warning' and 'failure' patients based on the ELN 2013 recommendations were assessed; cumulative MMR and MR(4.5) rates were 62.5% (n = 39) and 21.0% (n = 40), respectively, at 12 months. Twelve patients who showed a BCR-ABL transcript level >1% on the international scale did not achieve a MMR or discontinued dasatinib treatment because of insufficient effects. With regard to safety issues, grade 3/4 non-hematologic adverse events (AEs) were infrequent. CONCLUSIONS: Patients with non-optimal responses (who meet ELN 2013 warning and failure criteria) to imatinib should be switched quickly to dasatinib, which is less toxic in CML-CP patients, to improve their prognoses. A BCR-ABL1 IS of <1% at 3 months of dasatinib administration is a landmark for good therapeutic outcome.

CD4+ T cells in aged or thymectomized recipients of allogeneic stem cell transplantations.

BACKGROUND: CD4+CD25highFOXP3+ regulatory T (Treg) cells, which include thymus-derived and peripherally induced cells, play a central role in immune regulation, and are therefore crucial to prevent graft-versus-host disease (GVHD). The increasing use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for elderly patients with thymus regression, and our case of allo-HSCT shortly after total thymectomy, raised questions about the activity of thymus-derived Treg cells and peripherally induced Treg cells, which are otherwise indistinguishable. RESULTS: We found that despite pre-transplant thymectomy or older age, both naïve and effector Treg cells, as well as naïve and effector conventional T cells, proliferated in allo-HSCT recipients. Higher proportions of total Treg cells 1 month post allo-HSCT, and naïve Treg cells 1 year post allo-HSCT, appeared in patients achieving complete chimera without developing significant chronic GVHD, including our thymectomized patient, compared with patients who developed chronic GVHD. CONCLUSIONS: Treg cells that modulate human allogeneic immunity may arise peripherally as well as in the thymus of allo-HSCT recipients.

Peripheral blood progenitor cell collection by two programs for autologous and allogeneic transplantation.

BACKGROUND: In the Spectra apheresis instrument (Terumo BCT), both manual (Spectra-MNC) and automated (Spectra-Auto) programs have been widely used to collect peripheral blood progenitor cells (PBPCs). However, direct comparison of these programs remains extremely limited. STUDY DESIGN AND METHODS: We investigated 188 collections and products from autologous (patient) and allogeneic (donor) subjects and analyzed a subset of 89 allogeneic collections and products. Twenty-nine subjects who received apheresis for 2 consecutive days using both programs were also evaluated with a paired crossover comparison. RESULTS: The two programs processed similar volumes, but run time was longer with Spectra-Auto. Yield and efficiency of CD34+ cell collection were similar between these programs in the whole cohort, although white blood cell (WBC) and mononuclear cell (MNC) yields were higher with Spectra-MNC. In the allogeneic cohort, yield and efficiency of WBC collection were greater in Spectra-MNC. However, collected WBCs, MNCs, and CD34+ cells were similar between these programs in paired comparison. Regardless of program, preapheresis peripheral WBC, MNC, and CD34+ cell counts correlated with the number of cells collected. In contrast, preapheresis WBC counts in the whole cohort were negatively correlated with collection efficiencies of CD34+ cells in Spectra-MNC but not Spectra-Auto. The products collected using Spectra-MNC contained more contaminating platelets (PLTs) than Spectra-Auto, with a corresponding reduction in postdonation circulating PLTs. CONCLUSION: Spectra-MNC and Spectra-Auto showed distinct features that should be considered on a case-by-case basis. Similar investigations should be undertaken as new collection platforms are introduced.

Outcome after first relapse in adult patients with Philadelphia chromosome-negative acute lymphoblastic leukaemia.

To analyse the outcome of adult patients who developed a first relapse of acute lymphoblastic leukaemia (ALL), we collected the clinical data of 332 patients with Philadelphia-chromosome (Ph) negative ALL, aged 16-65 years, who relapsed after first complete remission (CR1) between 1998 and 2008 in 69 institutions all over Japan, including 58 patients who relapsed after allogeneic haematopoietic stem cell transplantation (Allo-HSCT) in CR1. The overall survival (OS) was 43·4% at 1 year, and 16·3% at 5 years from relapse in patients who received chemotherapy alone in CR1. Among patients who relapsed after chemotherapy alone in CR1, 123 (52·5%) achieved a second remission (CR2) following salvage chemotherapy, of whom 62 subsequently underwent Allo-HSCT during CR2. Allo-HSCT in CR2 was significantly associated with better OS. Moreover, the type of salvage chemotherapy influenced OS from relapse. A doxorubicin, vincristine, and predonisone-based (AdVP-type) regimen was related to better OS in patients with longer CR1 (more than 1 year), but was related to worse OS in patients with shorter CR1. In conclusion, the prognosis of patients with relapsed Ph-negative ALL is poor. Allo-HSCT after a first relapse could improve the prognosis. Selection of the optimal salvage chemotherapy might depend on the duration of CR1.

Performance characteristics of 5 numerical indexes in mixing test interpretation under coexistence of lupus anticoagulant and coagulation factor deficiency.

Background: The mixing test is useful to investigate the cause of unexpectedly prolonged activated partial thromboplastin time (APTT). Several indexes are available for distinguishing correction from non-correction (ie, factor deficiency from inhibitors), but their performance characteristics may differ because of their different formulas. Furthermore, it is unclear how each index performs under the coexistence of factor deficiency and inhibitors. Objectives: The objective of this study was to examine the differences in indexes, depending on factor VIII activity (FVIII:C) levels and lupus anticoagulant (LA) titers in test samples. Methods: APTT was measured in spiked samples with various FVIII:C levels and LA titers, normal pooled plasma (NPP), and their 4:1, 1:1, and 1:4 mixtures. The following 5 indexes were calculated: index of circulating anticoagulant, mixing test normalized ratio, 4:1 and 1:1 percent corrections, and an APTT difference between the 1:1 mixture and NPP. The samples with LA, showing correction, were measured for FVIII:C in a one-stage assay to check parallelism. Results: All indexes showed correction under FVIII deficiency and non-correction under higher LA titers. However, under lower LA titers, some indexes showed non-correction but others showed correction because of dilution effects and variations in formulas and/or sample mix ratios. The differences among the indexes were more pronounced under coexistent FVIII deficiency and LA, even though LA titers were equal in the tested samples; samples with lower FVIII:C showed correction, whereas those with normal FVIII:C showed non-correction. The samples tested for FVIII:C showed non-parallelism. Conclusion: Each index had different performance characteristics to LA samples, which were pronounced under low FVIII:C levels in test samples.

The suppressive effects of Mer inhibition on inflammatory responses in the pathogenesis of LPS-induced ALI/ARDS.

The pathogenesis of sepsis-induced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) has not yet been fully elucidated. Growth arrest-specific 6 (Gas6) has marked effects on hemostasis and reduces inflammation through its interaction with receptor tyrosine kinases of the TAM family: Tyro3, Axl, and Mer. Here, we found that plasma concentrations of Gas6 and soluble Mer were greater in patients with severe sepsis or septic ALI/ARDS compared with those in normal healthy donors. To determine whether the Gas6-Mer axis was critical in the pathogenesis of ALI/ARDS, we investigated the effects of intravenous administration of the selective Mer inhibitor UNC2250 on lipopolysaccharide (LPS)-induced ALI in mouse models subjected to inhalation of LPS. UNC2250 markedly inhibited the infiltration into the lungs of neutrophils and monocytes with increased amounts of Gas6 and Mer proteins, severe lung damage, and increased amounts of reactive oxygen species (ROS) in LPS-induced ALI in mice. In human pulmonary aortic endothelial cells, LPS induced decreases in the amounts of endothelial nitric oxide synthase, thrombomodulin, and vascular endothelial-cadherin, which was blocked by treatment with UNC2250. UNC2250 also inhibited the LPS-dependent increases in cell proliferation and enhanced apoptosis in HL-60 cells, a human neutrophil-like cell line, and RAW264.7 cells, a mouse monocyte/macrophage cell line. These data provide insights into the potential multiple beneficial effects of the Mer inhibitor UNC2250 as a therapeutic reagent to treat inflammatory responses in ALI/ARDS.

A Case of Acquired von Willebrand Syndrome Complicated by Acute Myelomonocytic Leukemia.

We here report a 21-year-old male who presented with acute myelomonocytic leukemia (AMML) associated with acquired von Willebrand syndrome (AVWS). To our knowledge, this is the first case of AVWS caused by AMML. In our case, following remission-induction chemotherapy combined with idarubicin and cytarabine, the patient showed remarkable improvement of bleeding symptoms due to AVWS. Moreover, after an allogeneic stem cell transplantation from a sibling donor, both AMML and AVWS maintain complete remission.

Myeloproliferative neoplasm-driving Calr frameshift promotes the development of pulmonary hypertension in mice.

Frameshifts in the Calreticulin (CALR) exon 9 provide a recurrent driver mutation of essential thrombocythemia (ET) and primary myelofibrosis among myeloproliferative neoplasms (MPNs). Here, we generated knock-in mice with murine Calr exon 9 mimicking the human CALR mutations, using the CRISPR-Cas9 method. Knock-in mice with del10 [Calrdel10/WT (wild-type) mice] exhibited an ET phenotype with increases of peripheral blood (PB) platelets and leukocytes, and accumulation of megakaryocytes in bone marrow (BM), while those with ins2 (Calrins2/WT mice) showed a slight splenic enlargement. Phosphorylated STAT3 (pSTAT3) was upregulated in BM cells of both knock-in mice. In BM transplantation (BMT) recipients from Calrdel10/WT mice, although PB cell counts were not different from those in BMT recipients from CalrWT/WT mice, Calrdel10/WT BM-derived macrophages exhibited elevations of pSTAT3 and Endothelin-1 levels. Strikingly, BMT recipients from Calrdel10/WT mice developed more severe pulmonary hypertension (PH)-which often arises as a comorbidity in patients with MPNs-than BMT recipients from CalrWT/WT mice, with pulmonary arterial remodeling accompanied by an accumulation of donor-derived macrophages in response to chronic hypoxia. In conclusion, our murine model with the frameshifted murine Calr presented an ET phenotype analogous to human MPNs in molecular mechanisms and cardiovascular complications such as PH.