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• Isogenic iPS cells with or without JAK2 V617F mutation were established. • JAK2 V617F mutation induced cell-autonomous erythropoiesis and megakaryopoiesis. • IFN-α inhibits erythropoiesis and megakaryopoiesis in cells with JAK2 V617F mutation. Identification and functional characterization of disease-associated genetic traits are crucial for understanding the pathogenesis of hematologic malignancies. Various in vitro and in vivo models, including cell lines, primary cells, and animal models, have been established to examine these genetic alterations. However, their nonphysiologic conditions, diverse genetic backgrounds, and species-specific differences often limit data interpretation. To evaluate somatic mutations in myeloproliferative neoplasms (MPNs), we used CRISPR/Cas9 combined with the piggyBac transposon system to establish isogenic induced pluripotent stem (iPS) cell lines with or without JAK2 V617F mutation, a driver mutation of MPNs. We induced hematopoietic stem/progenitor cells (HSPCs) from these iPS cells and observed phenotypic differences during hematopoiesis using fluorescence-activated cell sorting analysis. HSPCs with pathogenic mutations exhibited cell-autonomous erythropoiesis and megakaryopoiesis, which are hallmarks in the bone marrow of patients with MPNs. Furthermore, we used these HSPCs as a model to validate therapeutic compounds and showed that interferon alpha selectively inhibited erythropoiesis and megakaryopoiesis in mutant HSPCs. These results demonstrate that genome editing is feasible for establishing isogenic iPS cells, studying genetic elements to understand the pathogenesis of MPNs, and evaluating therapeutic compounds against MPNs. [ABSTRACT FROM AUTHOR]

Background: Acquired erythrocytosis can be classified into polycythemia vera (PV) and non‐neoplastic erythrocytosis (NNE). The vast majority of PV patients harbor JAK2 mutations, but differentiating JAK2 mutation‐negative PV from NNE is challenging due to a lack of definitive molecular markers. Methods: We studied the clinical features of 121 patients with erythrocytosis of which 47 (38.8%) were JAK2 mutation‐positive and also fulfilled the diagnostic criteria for PV, and 67 (55.4%) JAK2 mutation‐negative erythrocytosis patients who were diagnosed as NNE. Diagnosis was strictly based on driver mutation analysis and central pathology review. Results: No JAK2 mutation‐negative PV patients were found in our cohort. The NNE group showed significantly younger (p < 0.01) age with higher frequency of smoking (p < 0.001), alcohol consumption (p < 0.001), and diabetes mellitus (p < 0.05), whereas the PV group (n = 47) showed significantly higher white blood cell count, platelet count, and lactate dehydrogenase (p < 0.001). Although serum erythropoietin (EPO) levels were significantly higher in NNE compared to PV (p < 0.001), approximately 40% of the NNE patients had EPO levels below the lower range of normal, fulfilling a minor diagnostic criterion of PV and raising the possibility of PV misdiagnosis. Conclusion: Low EPO levels in JAK2 mutation‐negative erythrocytosis may not be a reliable diagnostic criterion for distinguishing PV from NNE. [ABSTRACT FROM AUTHOR]

• CREB3L1 expression in RBCs discriminates JAK2 exon12 and V617F mutations. • CREB3L1 is required for the erythrocytosis associated with EPOR hyperactivation. • CREB3L1 may be a potential therapeutic target for MPN. [Display omitted] CREB3L1 , a gene encoding the endoplasmic reticulum stress transducer, is specifically overexpressed in platelet RNA from patients with myeloproliferative neoplasms (MPNs). However, the pathophysiological roles of CREB3L1 overexpression remain unclear. In the present study, we aimed to study CREB3L1 messenger RNA (mRNA) expression in the red blood cells (RBCs) of patients with MPN and its role in erythrocytosis. Elevated expression of CREB3L1 was exclusively observed in the RBCs of patients with polycythemia vera (PV) harboring JAK2 exon 12 mutations, but not in those harboring JAK2 V617F mutation or control subjects. In erythropoiesis, CREB3L1 expression was sharply induced in erythroblasts of bone marrow cells collected from patients with JAK2 exon 12 mutation. This was also evident when erythropoiesis was induced in vitro using hematopoietic stem and progenitor cells (HSPCs) with JAK2 exon 12 mutation. Interestingly, overexpression of CREB3L1 in RBCs was observed in patients with reactive erythrocytosis whose serum erythropoietin (EPO) levels exceeded 100 mIU/mL. Elevated CREB3L1 expression was also observed in the erythroblasts of a patient with acute erythroid leukemia. EPO-dependent induction of CREB3L1 was evident in erythroblasts differentiated from HSPCs in vitro, regardless of driver mutation status or MPN pathogenesis. These data strongly suggest that CREB3L1 overexpression in RBCs is associated with hyperactivation of the EPO receptor and its downstream molecule, JAK2. Short hairpin RNA (shRNA) knockdown of CREB3L1 expression in HSPCs blocked erythroblast formation in vitro. These results suggest that CREB3L1 is required for erythropoiesis in the presence of JAK2 exon 12 mutation or high level of EPO, possibly by antagonizing cellular stress. [ABSTRACT FROM AUTHOR]

The lysyl oxidase gene inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162 amino acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibited the Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven breast tumor burden in a xenograft model. Since its mechanism of action is largely unknown, co-affinity-purification/mass spectrometry was performed and the “Cbl-interacting protein of 85-kDa” (CIN85) identified as an associating protein. CIN85 is an SH3-containing adapter protein that is overexpressed in invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3 ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to the AMAP1 complex where its ubiquitination activity is necessary for cancer cells to develop an invasive phenotype and to degrade the matrix. Direct interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation analysis of lysates from breast cancer cells and of purified expressed proteins. CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85 regions and deletion mutants of LOX-PP were prepared and used to map the sites of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl. Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the invasive phenotype of breast cancer cells, including their ability to degrade the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP interacts with CIN85 via a novel SH3-binding motif and this association reduces CIN85-promoted invasion by breast cancer cells. [ABSTRACT FROM AUTHOR]

The WBC count (C), Hb value (D), platelet count (E), and LDH level (F) for the patients classified based on driver mutation status are shown. Finally, in the Results, under the subheading 'Noncanonical JAK2 and MPL mutant exhibits wild-type-equivalent levels of STAT5 activation', "One such patient was found to harbor MPL S204F in the abovementioned screening of noncanonical JAK2 and MPL mutations." now reads "One such patient was found to harbor MPL S204F." NC-JAK2/MPL: noncanonical JAK2 and MPL mutation. [Extracted from the article]

Finally, in the Results, under the subheading 'Noncanonical JAK2 and MPL mutant exhibits wild-type-equivalent levels of STAT5 activation', "One such patient was found to harbor MPL S204F in the abovementioned screening of noncanonical JAK2 and MPL mutations." now reads "One such patient was found to harbor MPL S204F." Comparison of TPO (C) and IL-6 (D) concentrations in the serum among the patients with TN-ET, patients with ET harboring driver mutations, and healthy controls. Graph: Figure 1 Comparison of clinical parameters between the TN-ET patients and mutated ET patients. [Extracted from the article]

A subset of essential thrombocythemia (ET) cases are negative for disease-defining mutations on JAK2, MPL, and CALR and defined as triple negative (TN). The lack of recurrent mutations in TN-ET patients makes its pathogenesis ambiguous. Here, we screened 483 patients with suspected ET in a single institution, centrally reviewed bone marrow specimens, and identified 23 TN-ET patients. Analysis of clinical records revealed that TN-ET patients were mostly young female, without a history of thrombosis or progression to secondary myelofibrosis and leukemia. Sequencing analysis and human androgen receptor assays revealed that the majority of TN-ET patients exhibited polyclonal hematopoiesis, suggesting a possibility of reactive thrombocytosis in TN-ET. However, the serum levels of thrombopoietin (TPO) and interleukin-6 in TN-ET patients were not significantly different from those in ET patients with canonical mutations and healthy individuals. Rather, CD34-positive cells from TN-ET patients showed a capacity to form megakaryocytic colonies, even in the absence of TPO. No signs of thrombocytosis were observed before TN-ET development, denying the possibility of hereditary thrombocytosis in TN-ET. Overall, these findings indicate that TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and is paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis. [ABSTRACT FROM AUTHOR]

The MPN driver mutation, JAK2 exon 12, is rare in MPN and has been exclusively found in polycythaemia vera (PV) (Scott [6]), with no reports of this mutation in MDS/MPN-RS-T to date. We would like to emphasize the importance of analysing JAK2 exon 12 mutation in patients with suspected MDS/MPN-RS-T, but who are negative for JAK2 V617F, CALR exon 9 and MPL W515 mutations. In summary, we identified a patient with MDS/MPN-RS-T harboring SF3B1 and JAK2 exon 12 mutations. Bone marrow morphologies of the patient harboring JAK2 exon 12 mutation, representing features associated with MDS/MPN-RS-T.Figure S3. [Extracted from the article]

Objective: Prefibrotic/early primary myelofibrosis (pre‐PMF) and essential thrombocythemia (ET) exhibited different features of bone marrow; however, this is not always easy to judge objectively, making pathologists' distinction often suboptimal. In the WHO 2008 criteria, pre‐PMF was not defined as a subgroup of PMF; therefore, affected patients were at a higher risk of misdiagnosis with ET. In this study, we examined the prevalence of pre‐PMF patients among those previously diagnosed with ET in Japan. Method: We reviewed bone marrow specimens and clinical and molecular parameters of patients who were previously diagnosed with ET by the WHO 2008 criteria. Results: Among 107 ET patients, 13 patients were redefined as having pre‐PMF. Pre‐PMF patients exhibited a higher frequency of MPL mutation and increased platelet counts compared to true ET patients. Molecular analysis revealed the frequencies of high‐risk molecular mutations, such as ASXL1, EZH2, and SRSF2, were significantly increased in pre‐PMF patients than those in true ET patients. Conclusion: These results demonstrated the value of reexamining clinical records for patients diagnosed with ET by the WHO 2008 criteria and emphasized that adequate examinations of patients' bone marrow are crucial for an accurate diagnosis of pre‐PMF and ET. [ABSTRACT FROM AUTHOR]

Summary: Somatic mutations in the calreticulin (CALR) gene have been found in most patients with JAK2‐ and MPL‐unmutated Philadelphia chromosome‐negative myeloproliferative neoplasms (MPNs). It has recently been shown that mutant CALR constitutively activates the thrombopoietin receptor MPL and, thus, plays a causal role in the development of MPNs. However, the roles of mutant CALR in human haematopoietic cell differentiation remain predominantly elusive. To examine the impact of the 5‐base insertion mutant CALR gene (Ins5) on haematopoietic cell differentiation, we generated induced pluripotent stem cells from an essential thrombocythaemia (ET) patient harbouring a CALR‐Ins5 mutation and from a healthy individual (WT). Megakaryopoiesis was more prominent in Ins5‐haematopoietic progenitor cells (Ins5‐HPCs) than in WT‐HPCs, implying that the system recapitulates megakaryocytosis observed in the bone marrow of CALR‐mutant ET patients. Ins5‐HPCs exhibited elevated expression levels of GATA1 and GATA2, suggesting a premature commitment to megakaryocytic differentiation in progenitor cells. We also demonstrated that 3‐hydroxy anagrelide markedly perturbed megakaryopoiesis, but not erythropoiesis. Collectively, we established an in vitro model system that recapitulates megakaryopoiesis caused by mutant CALR. This system can be used to validate therapeutic compounds for MPN patients harbouring CALR mutations and in detailed studies on mutant CALR in human haematological cell differentiation. [ABSTRACT FROM AUTHOR]

Cyclic AMP-response element-binding protein 3-like 1 (CREB3L1) is a gene involved in the unfolded protein response (UPR). Recently, we demonstrated that CREB3L1 is specifically overexpressed in the platelets of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). In this study, we aimed to show the clinical and biological relevance of CREB3L1 in these hematological diseases. Overexpression of CREB3L1 was specific to platelets in MPNs and associated with a higher risk of thrombosis and fibrotic transformation in essential thrombocythemia (ET) and polycythemia vera (PV) cases, respectively. Furthermore, we found that UPR genes were downregulated in platelets of patients with ET and PV, which were more pronounced in patients harboring the JAK2 V617F mutation. However, CREB3L1 overexpression does not alter UPR gene expression or cell proliferation in UT-7/TPO/CALRm cells exogenously expressing mutated calreticulin and HEL cells harboring endogenous JAK2 V617F. Furthermore, CREB3L1 overexpression did not modulate sensitivity to endoplasmic reticulum stress in these cell lines. Taken together, our data show 1) a potential role of CREB3L1 expression in platelets as a new marker of high-risk MPNs and 2) an association between CREB3L1 overexpression and UPR gene downregulation in these patients' platelets, with CREB3L1 not altering UPR in our in vitro models and possibly further in vivo mechanisms being involved. • CREB3L1 expression is specific for MPN platelets. • CREB3L1 expression is associated with higher risk ET and PV. • UPR genes are downregulated in ET and PV platelets. • UPR genes expression is independent of CREB3L1 expression in cell line models. • CREB3L1 does not alter cell proliferation/ER stress sensitivity in cell line models. [ABSTRACT FROM AUTHOR]

Objective: The goal of this study was to identify a reliable biomarker for age-related infertility. Design: Laboratory study. Setting: ART laboratory. Patient(s): Patients undergoing intracytoplasmic sperm injection or IVF cycles. Intervention(s): Expression of Glutathione S-transferase (GST) mRNA and protein in mural and cumulus granulosa cells obtained from infertile patients were examined by reverse transcriptase-polymerase chain reaction and immunofluorescence. Main Outcome Measure(s): Correlation between the expression of GST theta 1 (GSTT1) in granulosa cells and oocyte quality was a main outcome measure. Result(s): Expression of GSTT1 in granulosa cells from male factor patients was positively correlated with age and negatively with cumulus-oocyte complex maturity. When samples with high and low GSTT1 in granulosa cells were extracted from the other infertility factors, cumulus-oocyte complex maturity in the high GSTT1 group was significantly lower than that in the low GSTT1 group (high: 27.2% vs. low: 51.3%). The developmental capacity of oocytes in the high GSTT1 group was likely to be lower (high: 26.4% vs. low: 43.9%). Up-regulation of GSTT1 during aging may be promoted by FSH and H2O2, determined by an in vitro model. Conclusion(s): GSTT1 is a good indicator for age-related infertility. [Copyright &y& Elsevier]