CREB3L1 overexpression as a potential diagnostic marker of Philadelphia chromosome-negative myeloproliferative neoplasms.

Discrimination of Philadelphia-negative myeloproliferative neoplasms (Ph-MPNs) from reactive hypercytosis and myelofibrosis requires a constellation of testing including driver mutation analysis and bone marrow biopsies. We searched for a biomarker that can more easily distinguish Ph-MPNs from reactive hypercytosis and myelofibrosis by using RNA-seq analysis utilizing platelet-rich plasma (PRP)-derived RNAs from patients with essential thrombocythemia (ET) and reactive thrombocytosis, and CREB3L1 was found to have an extremely high impact in discriminating the two disorders. To validate and further explore the result, expression levels of CREB3L1 in PRP were quantified by reverse-transcription quantitative PCR and compared among patients with ET, other Ph-MPNs, chronic myeloid leukemia (CML), and reactive hypercytosis and myelofibrosis. A CREB3L1 expression cutoff value determined based on PRP of 18 healthy volunteers accurately discriminated 150 driver mutation-positive Ph-MPNs from other entities (71 reactive hypercytosis and myelofibrosis, 6 CML, and 18 healthy volunteers) and showed both sensitivity and specificity of 1.0000. Importantly, CREB3L1 expression levels were significantly higher in ET compared with reactive thrombocytosis (P < .0001), and polycythemia vera compared with reactive erythrocytosis (P < .0001). Pathology-affirmed triple-negative ET (TN-ET) patients were divided into a high- and low-CREB3L1-expression group, and some patients in the low-expression group achieved a spontaneous remission during the clinical course. In conclusion, CREB3L1 analysis has the potential to single-handedly discriminate driver mutation-positive Ph-MPNs from reactive hypercytosis and myelofibrosis, and also may identify a subgroup within TN-ET showing distinct clinical features including spontaneous remission.

Clinical and molecular features of patients with prefibrotic primary myelofibrosis previously diagnosed as having essential thrombocythemia in Japan.

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.

Histone Acetyltransferase p300/CREB-binding Protein-associated Factor (PCAF) Is Required for All-trans-retinoic Acid-induced Granulocytic Differentiation in Leukemia Cells.

Differentiation therapy with all-trans-retinoic acid (ATRA) improves the treatment outcome of acute promyelocytic leukemia (APL); however, the molecular mechanism by which ATRA induces granulocytic differentiation remains unclear. We previously reported that the inhibition of the NAD-dependent histone deacetylase (HDAC) SIRT2 induces granulocytic differentiation in leukemia cells, suggesting the involvement of protein acetylation in ATRA-induced leukemia cell differentiation. Herein, we show that p300/CREB-binding protein-associated factor (PCAF), a histone acetyltransferase (HAT), is a prerequisite for ATRA-induced granulocytic differentiation in leukemia cells. We found that PCAF expression was markedly increased in leukemia cell lines (NB4 and HL-60) and primary APL cells during ATRA-induced granulocytic differentiation. Consistent with these results, the expression of PCAF was markedly up-regulated in the bone marrow cells of APL patients who received ATRA-containing chemotherapy. The knockdown of PCAF inhibited ATRA-induced granulocytic differentiation in leukemia cell lines and primary APL cells. Conversely, the overexpression of PCAF induced the expression of the granulocytic differentiation marker CD11b at the mRNA level. Acetylome analysis identified the acetylated proteins after ATRA treatment, and we found that histone H3, a known PCAF acetylation substrate, was preferentially acetylated by the ATRA treatment. Furthermore, we have demonstrated that PCAF is required for the acetylation of histone H3 on the promoter of ATRA target genes, such as CCL2 and FGR, and for the expression of these genes in ATRA-treated leukemia cells. These results strongly support our hypothesis that PCAF is induced and activated by ATRA, and the subsequent acetylation of PCAF substrates promotes granulocytic differentiation in leukemia cells. Targeting PCAF and its downstream acetylation targets could serve as a novel therapeutic strategy to overcome all subtypes of AML.

Skewed megakaryopoiesis in human induced pluripotent stem cell-derived haematopoietic progenitor cells harbouring calreticulin mutations.

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.

Target antigen density governs the efficacy of anti-CD20-CD28-CD3 ζ chimeric antigen receptor-modified effector CD8+ T cells.

The effectiveness of chimeric Ag receptor (CAR)-transduced T (CAR-T) cells has been attributed to supraphysiological signaling through CARs. Second- and later-generation CARs simultaneously transmit costimulatory signals with CD3ζ signals upon ligation, but may lead to severe adverse effects owing to the recognition of minimal Ag expression outside the target tumor. Currently, the threshold target Ag density for CAR-T cell lysis and further activation, including cytokine production, has not yet been investigated in detail. Therefore, we determined the threshold target Ag density required to induce CAR-T cell responses using novel anti-CD20 CAR-T cells with a CD28 intracellular domain and a CD20-transduced CEM cell model. The newly developed CD20CAR-T cells demonstrated Ag-specific lysis and cytokine secretion, which was a reasonable level as a second-generation CAR. For lytic activity, the threshold Ag density was determined to be ∼200 molecules per target cell, whereas the Ag density required for cytokine production of CAR-T cells was ∼10-fold higher, at a few thousand per target cell. CD20CAR-T cells responded efficiently to CD20-downregulated lymphoma and leukemia targets, including rituximab- or ofatumumab-refractory primary chronic lymphocytic leukemia cells. Despite the potential influence of the structure, localization, and binding affinity of the CAR/Ag, the threshold determined may be used for target Ag selection. An Ag density below the threshold may not result in adverse effects, whereas that above the threshold may be sufficient for practical effectiveness. CD20CAR-T cells also demonstrated significant lytic activity against CD20-downregulated tumor cells and may exhibit effectiveness for CD20-positive lymphoid malignancies.

Environmental carcinogenesis - 100th anniversary of creating cancer.

Asbestos is an environmental carcinogen, and asbestos-related diseases represent a global-scale environmental issue. Mesothelioma is an aggressive, malignant tumor that initially progresses along the surfaces of the pleura and peritoneum that is chiefly attributed to asbestos exposure. X-rays are commonly used for tumor screening in populations at risk for developing this cancer. We previously reported that the N-terminal of mesothelin may be a useful blood marker for early diagnosis method for mesothelioma and since then developed an N-terminal of mesothelin ELISA kit in collaboration with IBL Co., Ltd. and confirmed its utility as a diagnostic system for mesothelioma. Recently, we performed a large-scale research screening for mesothelioma and showed that it is a good model for early diagnosis in at-risk populations. The year 2015 is the 100th anniversary of Yamagiwa's great work on coaltar-induced carcinogenesis by formative stimulation in 1915 and the 10th year since 2005, "Kubota shock", people recognized that asbestos induces mesothelioma. We dedicate this review to this memorial year for environmental carcinogenesis.

Tumor suppressor function of Liver kinase B1 (Lkb1) is linked to regulation of epithelial integrity.

Although loss of epithelial integrity is a hallmark of advanced cancer, it remains poorly understood whether genetic alterations corrupting this integrity causally facilitate tumorigenesis. We show that conditional deletion of tumor suppressor gene Lkb1 (Par-4) in the mammary gland compromises epithelial integrity manifested by mislocalization of cell polarity markers, lateralization of tight junctions, deterioration of desmosomes and basement membrane (BM), and hyperbranching of the mammary ductal tree. We identify the desmosomal BM remodelling serine protease Hepsin as a key factor mediating Lkb1 loss-induced structural alterations in mammary epithelium and BM fragmentation. Although loss of Lkb1 alone does not promote mammary tumorigenesis, combination of Lkb1 deficiency with oncogenic c-Myc leads to dramatic acceleration in tumor formation. The results coupling Lkb1 loss-mediated epithelial integrity defects to mislocalization of serine protease Hepsin and to oncogenic synergy with c-Myc imply that Lkb1 loss facilitates oncogenic proliferation by releasing epithelial cells from structural BM boundaries.

Establishment of isogenic induced pluripotent stem cells with or without pathogenic mutation for understanding the pathogenesis of myeloproliferative neoplasms.

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 JAK2V617F 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.

Clinical features of acquired erythrocytosis: Low levels of serum erythropoietin in a subset of non-neoplastic erythrocytosis patients.

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.

Clinical and biological relevance of CREB3L1 in Philadelphia chromosome-negative myeloproliferative neoplasms.

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.

Correlation between serum albumin and serum zinc in malignant lymphoma.

Objectives: Zinc (Zn) is a cofactor for more than 200 enzymes within the human body. Zn deficiency can result in cell-mediated immune dysfunction. Furthermore, serum Zn levels have been reported to be associated with nutritional status, but this association has not been clarified in malignant lymphoma. This study aimed to examine the deficiency of serum Zn levels and clarify the factors that are correlated with serum Zn in malignant lymphoma. Methods: Initial malignant lymphoma was diagnosed in patients at Fujita Health University Hospital between April 2011 and March 2019. Based on the serum Zn levels, the study population was divided into "deficient" and "low or normal". For the serum Zn levels of patients undergoing pre-chemotherapy, laboratory parameters and nutritional factors were included. We compared these factors between the abovementioned two groups, and the serum Zn levels with its correlation factors were investigated. Results: A total of 77 patients (Deficient group, n=20 and Low or Normal group, n=57) were enrolled. Histology, hemoglobin, serum albumin levels, Glasgow Prognostic Score (GPS), neutrophile-lymphocyte ratio (NLR), prognostic nutrition index (PNI) and Controlling Nutritional Status (CONUT) were significantly different between the two groups. Of these parameters, only serum albumin level was significantly associated with serum Zn level (p=0.0024; estimated regression coefficient, 9.51; adjusted coefficient of determination, 0.28). Conclusions: Poor nutritional status at the initial diagnosis may have affected Zn deficiency in initial malignant lymphoma.

Eliminating chronic myeloid leukemia stem cells by IRAK1/4 inhibitors.

Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are quiescent, insensitive to BCR-ABL1 tyrosine kinase inhibitors (TKIs) and responsible for CML relapse. Therefore, eradicating quiescent CML LSCs is a major goal in CML therapy. Here, using a G0 marker (G0M), we narrow down CML LSCs as G0M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis reveals NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by inhibitors of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4 inhibitors) together with imatinib eliminates mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuate PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eliminates CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors can eliminate CML LSCs through inhibiting NF-κB activity and reducing PD-L1 expression. Collectively, the combination of TKIs and IRAK1/4 inhibitors is an attractive strategy to achieve a radical cure of CML.

A marine sponge-derived lectin reveals hidden pathway for thrombopoietin receptor activation.

N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that traffics to the cell surface and constitutively activates the receptor. However, the molecular basis for this mechanism is elusive because oncogenic activation occurs only in the cell-intrinsic complex and is thus cannot be replicated with external agonists. Here, we describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC), a homodimerized lectin with calcium-dependent fucose-binding properties. In-depth characterization of lectin-induced activation showed that, similar to oncogenic activation, sugar chain-mediated activation persists due to limited receptor internalization. The strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. Overall, the existence of sugar-mediated MPL activation, in which the mode of activation is different from the original ligand, suggests that receptor activation is unpredictably diverse in living organisms.

MPL overexpression induces a high level of mutant-CALR/MPL complex: a novel mechanism of ruxolitinib resistance in myeloproliferative neoplasms with CALR mutations.

Ruxolitinib (RUX), a JAK1/2-inhibitor, is effective for myeloproliferative neoplasm (MPN) with both JAK2V617 F and calreticulin (CALR) mutations. However, many MPN patients develop resistance to RUX. Although mechanisms of RUX-resistance in cells with JAK2V617 F have already been characterized, those in cells with CALR mutations remain to be elucidated. In this study, we established RUX-resistant human cell lines with CALR mutations and characterized mechanisms of RUX-resistance. Here, we found that RUX-resistant cells had high levels of MPL transcripts, overexpression of both MPL and JAK2, and increased phosphorylation of JAK2 and STAT5. We also found that mature MPL proteins were more stable in RUX-resistant cells. Knockdown of MPL in RUX-resistant cells by shRNAs decreased JAK/STAT signaling. Immunoprecipitation assays showed that binding of mutant CALR to MPL was increased in RUX-resistant cells. Reduction of mutated CALR decreased proliferation of the resistant cells. When resistant cells were cultured in the absence of RUX, the RUX-resistance was reversed, with reduction of the mutant-CALR/MPL complex. In conclusion, MPL overexpression induces higher levels of a mutant-CALR/MPL complex, which may cause RUX-resistance in cells with CALR mutations. This mechanism may be a new therapeutic target to overcome RUX-resistance.

Cell-autonomous megakaryopoiesis associated with polyclonal hematopoiesis in triple-negative essential thrombocythemia.

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.

In silico-labeled ghost cytometry.

Characterization and isolation of a large population of cells are indispensable procedures in biological sciences. Flow cytometry is one of the standards that offers a method to characterize and isolate cells at high throughput. When performing flow cytometry, cells are molecularly stained with fluorescent labels to adopt biomolecular specificity which is essential for characterizing cells. However, molecular staining is costly and its chemical toxicity can cause side effects to the cells which becomes a critical issue when the cells are used downstream as medical products or for further analysis. Here, we introduce a high-throughput stain-free flow cytometry called in silico-labeled ghost cytometry which characterizes and sorts cells using machine-predicted labels. Instead of detecting molecular stains, we use machine learning to derive the molecular labels from compressive data obtained with diffractive and scattering imaging methods. By directly using the compressive 'imaging' data, our system can accurately assign the designated label to each cell in real time and perform sorting based on this judgment. With this method, we were able to distinguish different cell states, cell types derived from human induced pluripotent stem (iPS) cells, and subtypes of peripheral white blood cells using only stain-free modalities. Our method will find applications in cell manufacturing for regenerative medicine as well as in cell-based medical diagnostic assays in which fluorescence labeling of the cells is undesirable.

Age-associated changes in the subcellular localization of phosphorylated p38 MAPK in human granulosa cells.

p38 MAPK (p38) plays pivotal roles in aging and reproductive physiology. Nevertheless, involvement of p38 in female reproductive aging is uncertain. To improve knowledge of the role of p38 in age-associated reproductive failure, the expression and subcellular localization of phosphorylated p38 was investigated in human granulosa cells. p38 was 7-fold more activated in cells from older subjects than in those from younger subjects. Similar results were obtained in human granulosa-like KGN cells treated with hydrogen peroxide (H(2)O(2)). Interestingly, phosphorylated p38 was detected in the nucleus less frequently in older cells than in younger cells (Younger: 58.6%; Older: 29.8%, P< 0.01). Similarly cytoplasmic localization of phosphorylated p38 in KGN cells was observed after treatment with H(2)O(2). The activation and cytoplasmic localization of p38 in H(2)O(2)-treated KGN cells were blocked by N-acetylcysteine and SB203580. Although the p38 activators, FSH and tumor necrosis factor-α, induced a similar localization of phosphorylated p38 in KGN cells, the expression and localization patterns of p38 were distinct from those in older granulosa cells and H(2)O(2)-treated KGN cells. These results indicate that the characteristic localization of p38 in older granulosa cells is induced by oxidative stress.

Mutant calreticulin interacts with MPL in the secretion pathway for activation on the cell surface.

Studies have shown that mutant calreticulin (CALR) constitutively activates the thrombopoietin (TPO) receptor MPL and thus plays a causal role in the development of myeloproliferative neoplasms (MPNs). To further elucidate the molecular mechanism by which mutant CALR promotes MPN development, we studied the subcellular localization of mutant CALR and its importance for the oncogenic properties of mutant CALR. Here, mutant CALR accumulated in the Golgi apparatus, and its entrance into the secretion pathway and capacity to interact with N-glycan were required for its oncogenic capacity via the constitutive activation of MPL. Mutant CALR-dependent MPL activation was resistant to blockade of intracellular protein trafficking, suggesting that MPL is activated before reaching the cell surface. However, removal of MPL from the cell surface with trypsin shut down downstream activation, implying that the surface localization of MPL is required for mutant CALR-dependent activation. Furthermore, we found that mutant CALR and MPL interact on the cell surface. Based on these findings, we propose a model in which mutant CALR induces MPL activation on the cell surface to promote MPN development.