Phenotypic modulation of smooth muscle cells is a hallmark of disease. The associated expansion of endoplasmic reticulum (ER) volume remains unexplained. Thrombospondin-4 was recently found to promote ATF6α activation leading to ER expansion. Using bladder outlet obstruction as a paradigm for phenotypic modulation, we tested if thrombospondin-4 is induced in association with ATF6α activation and ER expansion. Thrombospondin-4 was induced and ATF6α was activated after outlet obstruction in rodents. Increased abundance of spliced of Xbp1, another ER-stress sensor, and induction of Atf4 and Creb3l2 was also seen. Downstream of ATF6α, Calr, Manf, Sdf2l1 and Pdi increased as did ER size, whereas contractile markers were reduced. Overexpression of ATF6α, but not of thrombospondin-4, increased Calr, Manf, Sdf2l1 and Pdi and caused ER expansion, but the contractile markers were inert. Knockout of thrombospondin-4 neither affected bladder growth nor expression of ATF6α target genes, and repression of contractile markers was the same, even if ATF6α activation was curtailed. Increases of Xbp1s, Atf4 and Creb3l2 were similar. Our findings demonstrate reciprocal regulation of the unfolded protein response, including ATF6α activation and ER expansion, and reduced contractile differentiation in bladder outlet obstruction occurring independently of thrombospondin-4, which however is a sensitive indicator of obstruction.

Metabolic syndrome is characterized by central obesity, insulin resistance, elevated blood pressure, and dyslipidemia. Metabolic syndrome is a significant risk factor for several common cancers (e.g., liver, colorectal, breast, pancreas). Pharmacologic treatments used for the components of the metabolic syndrome appear to be insufficient to control cancer development in subjects with metabolic syndrome. Murine models showed that cancer has the slowest progression when there is no food consumption during the daily activity phase. Intermittent fasting from dawn to sunset is a form of fasting practiced during human activity hours. To test the anticancer effect of intermittent fasting from dawn to sunset in metabolic syndrome, we conducted a pilot study in 14 subjects with metabolic syndrome who fasted (no eating or drinking) from dawn to sunset for more than 14 h daily for four consecutive weeks. We collected serum samples before 4-week intermittent fasting, at the end of 4th week during 4-week intermittent fasting and 1 week after 4-week intermittent fasting. We performed serum proteomic analysis using nano ultra-high performance liquid chromatography-tandem mass spectrometry. We found a significant fold increase in the levels of several tumor suppressor and DNA repair gene protein products (GP)s at the end of 4th week during 4-week intermittent fasting (CALU, INTS6, KIT, CROCC, PIGR), and 1 week after 4-week intermittent fasting (CALU, CALR, IGFBP4, SEMA4B) compared with the levels before 4-week intermittent fasting. We also found a significant reduction in the levels of tumor promoter GPs at the end of 4th week during 4-week intermittent fasting (POLK, CD109, CAMP, NIFK, SRGN), and 1 week after 4-week intermittent fasting (CAMP, PLAC1) compared with the levels before 4-week intermittent fasting. Fasting from dawn to sunset for four weeks also induced an anti-diabetes proteome response by upregulating the key regulatory proteins of insulin signaling at the end of 4th week during 4-week intermittent fasting (VPS8, POLRMT, IGFBP-5) and 1 week after 4-week intermittent fasting (PRKCSH), and an anti-aging proteome response by upregulating H2B histone proteins 1 week after 4-week intermittent fasting. Subjects had a significant reduction in body mass index, waist circumference, and improvement in blood pressure that co-occurred with the anticancer, anti-diabetes, and anti-aging serum proteome response. These findings suggest that intermittent fasting from dawn to sunset actively modulates the respective genes and can be an adjunct treatment in metabolic syndrome. Further studies are needed to test the intermittent fasting from dawn to sunset in the prevention and treatment of metabolic syndrome-induced cancers.

OBJECTIVE

In this study we hypothesized that the mRNA vector Staufen mediates RNA relocalization during meiotic maturation, and by virtue of its interactions with endoplasmic reticulum, provides a possible mechanism by which protein synthesis is regulated.

METHODS

We assessed the expression of staufen (STAU) and calreticulin (CALR), the latter adopted as a marker of the endoplasmic reticulum, in human oocytes at different stages of maturation: GV, metaphase MI and MII. Oocytes were subjected to polymerase chain reaction in order to investigate the expression of STAU and CALR. The corresponding protein products were identified by immunofluorescence and confocal laser scanning microscopy.

RESULTS

STAU and CALR were constantly expressed and selectively localized during oocyte maturation. At the GV stage the both proteins displayed a dispersed distribution localization throughout the cytoplasm. Progressing to the MII stage, STAU tended to compartmentalize towards the cortical area of the oocyte clustering in granules of larger sizes. At the MII stage, CALR assumed a pattern reminiscent and possibly coincident with the position of the meiotic spindle.

CONCLUSIONS

The changing pattern of STAU distribution during meiotic maturation of human oocytes implicates a novel mechanism for the regulation of protein synthesis based on mRNA localization. Moreover, the unique disposition of CALR at the MII spindle uncovers a physical interaction with endoplasmic reticulum that may mediate cytoskeletal remodelling during oocyte maturation.

Calreticulin (CALR) is a Ca2+-binding protein that shuttles among cellular compartments with proteins bound to its N/P domains. The knowledge that activation of the human erythropoietin receptor induces Ca2+ fluxes prompted us to investigate the role of CALR in human erythropoiesis. As shown by Western blot analysis, erythroblasts generated in vitro from normal sources and JAK2V617F polycythemia vera (PV) patients expressed robust levels of CALR. However, Ca2+ regulated CALR conformation only in normal cells. Normal erythroblasts expressed mostly the N-terminal domain of CALR (N-CALR) on their cell surface (as shown by flow cytometry) and C-terminal domain (C-CALR) in their cytoplasm (as shown by confocal microscopy) and expression of both epitopes decreased with maturation. In the proerythroblast (proEry) cytoplasm, C-CALR was associated with the glucocorticoid receptor (GR), which initiated the stress response. In these cells, Ca2+ deprivation and inhibition of nuclear export increased GR nuclear localization while decreasing cytoplasmic detection of C-CALR and C-CALR/GR association and proliferation in response to the GR agonist dexamethasone (Dex). C-CALR/GR association and Dex responsiveness were instead increased by Ca2+ and erythropoietin. In contrast, JAK2V617F proErys expressed normal cell-surface levels of N-CALR but barely detectable cytoplasmic levels of C-CALR. These cells contained GR mainly in the nucleus and were Dex unresponsive. Ruxolitinib rescued cytoplasmic detection of C-CALR, C-CALR/GR association, and Dex responsiveness in JAK2V617F proErys and its effects were antagonized by nuclear export and Ca2+ flux inhibitors. These results indicates that Ca2+-induced conformational changes of CALR regulate nuclear export of GR in normal erythroblasts and that JAK2V617F deregulates this function in PV.

OBJECTIVE

In the last decade, a number of genes have been reported to be recurrently associated with myeloid malignancies. While some mutations are easily detectable by conventional molecular genetics methods, other mutations are more difficult to screen because of lower frequency and being scattered along large genomic ranges. However, newly developed approaches for next-generation sequencing provide an affordable solution for targeted multiplex resequencing of up to several hundreds of amplicons. Here, we aimed to develop and validate a novel custom panel for targeted resequencing of myeloid malignancy samples using the Ion PGM(™) System (Ion Torrent, Paisley, UK).

METHODS

We designed a pool of 424 primers for the amplification of 212 amplicons covering 99.46 % of the exonic regions of nine human genes as follows: ASXL1, EZH2, CALR, RUNX1, SETBP1, SF3B1, SRSF2, TET2, and U2AF1. Initial testing of the panel performance was performed on an Ion PGM(™) machine using PGM(™) 316 v2 chips on 16 DNA samples from patients with myeloid malignancies. Sequence alignment, variant calling, and annotation were performed using Ion Reporter software.

RESULTS

We identified a total of 14 nonsynonymous somatic coding variants in seven samples affecting six of the genes in the panel (ASXL1, CALR, RUNX1, SRSF2, TET2, and U2AF1). Notably, three of the identified mutations were not present in the Cosmic v.67 release.

CONCLUSIONS

This proof-of-concept study confirms the feasibility of Ion Torrent systems for resequencing of clinically relevant mutations in myeloid malignancies. It can be particularly useful in cases without the most frequent clonal markers.

OBJECTIVE

To validate a diagnostic assay for detecting CALR mutations in the clinical setting.

METHODS

Traditional polymerase chain reaction (PCR) was performed on DNA previously extracted from 60 specimens (30 bone marrow aspirates [BMAs] and 30 peripheral blood [PB] samples) from 55 patients. Nearly all reported CALR mutations are insertions or deletions in exon 9. Therefore, we performed amplicon sizing by capillary electrophoresis and fragment length analysis (FLA) to determine mutation status. Mutations were confirmed by Sanger sequencing.

RESULTS

Fourteen samples from 10 patients with JAK2 and MPL wild-type myeloproliferative neoplasms were positive for CALR mutation. Detected mutations included a 52-base pair (bp) deletion (n = 6), a 5-bp insertion (n = 2), a 31-bp deletion (n = 1), and a 61-bp deletion (n = 1). Sanger sequencing of 15 samples showed 100% concordance. Matched patient PB and BMA samples (n = 5) harbored identical mutations, and samples run multiple times (n = 8) showed 100% reproducibility.

CONCLUSIONS

We conclude that CALR mutations may be quickly and accurately detected by FLA of PCR amplicons by capillary electrophoresis. These methods are routine procedures for most molecular laboratories and should allow for straightforward incorporation of the CALR assay into the clinical diagnostic testing menu.

BACKGROUND

Next to janus kinase 2 (JAK2) and myeloproliferative leukemia protein, calreticulin (CALR) is a recently discovered mutation present in>> 20% of patients diagnosed with primary myelofibrosis (PMF).

METHODS

Six studies published from December 2013 to December 2014 met the inclusion criteria for the present meta-analysis: 2 of an Asian and 4 of a non-Asian population. We assessed the biologic characteristics at diagnosis and investigated overall survival. The analyses were stratified by ethnic origin (Asian vs. non-Asian).

RESULTS

A total of 816 patients with the JAK2 mutation and 307 patients with the CALR mutation were included. The patients with the JAK2 mutation were older than those with the CALR mutation, and no statistically significant difference was noted in the gender distribution of the patients with PMF with the JAK2 versus CALR mutation. Patients with JAK2-mutated PMF had a higher white blood cell count, but no statistically significant evidence was found for a difference in the platelet count or hemoglobin level. No difference was found in thrombosis risk or acute leukemic transformation in those 2 populations. Major differences were found between the Asian and non-Asian populations. The difference in characteristics between JAK2 and CALR was larger in the Asian population than in the non-Asian population (P = .007). Also, in the non-Asian population, those with JAK2 mutation had lower platelet counts than the Asians (P = .06). In the non-Asian population, the patients diagnosed with JAK2-positive PMF had worse overall survival than the patients with CALR-positive PMF, with a combined hazard ratio of 2.43 (95% confidence interval, 1.83-3.22).

CONCLUSIONS

The results of the present meta-analysis have confirmed the role of the CALR mutation in the diagnosis of, and as a prognostic tool for, PMF. Our results suggest that patients with the CALR mutation will have better overall survival than patients with the JAK2 mutation in a non-Asian population.

Ursolic acid is a key active compound present in many medicinal herbs that have been widely used in traditional Chinese medicine for the clinical treatment of various cancers. However, the precise mechanisms of its antitumor activity have been poorly understood. To identify the cellular targets of ursolic acid, two-dimensional gel electrophoresis combined with mass spectrometry was performed in this study, which identified 15 proteins with significantly altered levels in protein expression. This demonstrated that ursolic acid-induced cytotoxicity in colorectal cancer cells involves dysregulation in protein folding, signal transduction, cell proliferation, cell cycle, and apoptosis. Corresponding protein regulation was also confirmed by Western blotting. Furthermore, the study of functional association between these 15 proteins revealed that 10 were closely related in a protein-protein interaction network, whereby the proteins either had a direct interaction with each other or were associated via only one intermediary protein. In this instance, the ATP5B/CALR/HSP90B1/HSPB1/HSPD1-signaling network was revealed as the predominant target which was associated with the majority of the observed protein-protein interactions. As a result, the identified targets may be useful in explaining the anticancer mechanisms of ursolic acid and as potential targets for colorectal cancer therapy.

Myeloproliferative neoplasms (MPNs) are clinically characterized by the chronic overproduction of differentiated peripheral blood cells and the gradual expansion of malignant intramedullary/extramedullary hematopoiesis. In MPNs mutations in JAK2 MPL or CALR are detected mutually exclusive in more than 90% of cases [1,2]. Mutations in them lead to the abnormal activation of JAK/STAT signaling and the autonomous growth of differentiated cells therefore they are considered as "driver" gene mutations. In addition to the above driver gene mutations mutations in epigenetic regulators such as TET2 DNMT3A ASXL1 EZH2 or IDH1/2 are detected in about 5%-30% of cases respectively [3]. Mutations in TET2 DNMT3A EZH2 or IDH1/2 commonly confer the increased self-renewal capacity on normal hematopoietic stem cells (HSCs) but they do not lead to the autonomous growth of differentiated cells and only exhibit subtle clinical phenotypes [4,6-8,5]. It was unclear how mutations in such epigenetic regulators influenced abnormal HSCs with driver gene mutations how they influenced the disease phenotype or whether a single driver gene mutation was sufficient for the initiation of human MPNs. Therefore we focused on JAK2V617F and loss of TET2-the former as a representative of driver gene mutations and the latter as a representative of mutations in epigenetic regulators-and examined the influence of single or double mutations on HSCs (Lineage(-)Sca-1(+)c-Kit(+) cells (LSKs)) by functional analyses and microarray whole-genome expression analyses [9]. Gene expression profiling showed that the HSC fingerprint genes [10] was statistically equally enriched in TET2-knockdown-LSKs but negatively enriched in JAK2V617F-LSKs compared to that in wild-type-LSKs. Double-mutant-LSKs showed the same tendency as JAK2V617F-LSKs in terms of their HSC fingerprint genes but the expression of individual genes differed between the two groups. Among 245 HSC fingerprint genes 100 were more highly expressed in double-mutant-LSKs than in JAK2V617F-LSKs. These altered gene expressions might partly explain the mechanisms of initiation and progression of MPNs which was observed in the functional analyses [9]. Here we describe gene expression profiles deposited at the Gene Expression Omnibus (GEO) under the accession number GSE62302 including experimental methods and quality control analyses.

Schisandrin A and B (Sch A and B) are the main effective components extracted from the oriental medicine Schisandra chinensis which is traditionally used to enhance mental and intellectual function. Although their neuroprotective effects have been demonstrated, their influences on neurogenesis are still unknown. In the brain, new neural cells born in the subventricular zone (SVZ) next to the lateral ventricles migrate along the rostral migratory stream (RMS) to the olfactory bulb (OB). To investigate the effects of Sch A and B on neurogenesis in the SVZ-RMS-OB system, Sch A and B were intragastrically administrated at dosages of 1, 10 and 20 mg/kg d respectively. The dose of 10 mg/kg d was selected for further analysis based on the preliminary analysis. In the SVZ, significant increases of phosphohistone H3 positive proliferating cells and the intensity of glial fibrillary acidic protein (GFAP+) cells were noticed in Sch B group. In the RMS, Sch A treatment augmented the intensity of doublecortin positive neuroblasts. In the OB, Sch A decreased tyrosine hydroxylase cells and Calbindin (CalB+) cells, while Sch B increased CalB+ cells and Calretinin (CalR+) cells. These results suggest that Sch B stimulates SVZ proliferation by enhancing GFAP+ cells and improves the survival of OB interneurons, while Sch A promotes neuroblast formation in the RMS but impairs the survival of OB interneurons. The present study provides the first evidence that Sch B exerts neuroprotective functions by enhancing neurogenesis, but Sch A mainly negatively regulates neurogenesis, in the adult SVZ-RMS-OB system.

Matrix metalloproteinase-9 (MMP-9) is a secreted endoproteinase with a critical role in the regulation of the extracellular matrix and proteolytic activation of signaling molecules. Human (h)MMP-9 has two well-defined N-glycosylation sites at residues N38 and N120; however, their role has remained mostly unexplored partly because expression of the N-glycosylation-deficient N38S has been difficult due to a recently discovered single nucleotide polymorphism-dependent miRNA-mediated inhibitory mechanism. hMMP-9 cDNA encoding amino acid substitutions at residues 38 (modified-S38, mS38) or 120 (N120S) were created in the background of a miRNA-binding site disrupted template and expressed by transient transfection. hMMP-9 harboring a single mS38 replacement secreted well, whereas N120S, or a double mS38/N120S hMMP-9 demonstrated much reduced secretion. Imaging indicated endoplasmic reticulum (ER) retention of the non-secreted variants and co-immunoprecipitation confirmed an enhanced strong interaction between the non-secreted hMMP-9 and the ER-resident protein calreticulin (CALR). Removal of N-glycosylation at residue 38 revealed an amino acid-dependent strong interaction with CALR likely preventing unloading of the misfolded protein from the ER chaperone down the normal secretory pathway. As with other glycoproteins, N-glycosylation strongly regulates hMMP-9 secretion. This is mediated, however, through a novel mechanism of cloaking an N-glycosylation-independent strong interaction with the ER-resident CALR.

CALR mutations are detected in about 50% of persons of predominately European descent with essential thrombocythemia (ET) or primary myelofibrosis (PMF) with wild-type alleles of JAK2 and MPL. We studied 1088 Chinese with diverse myeloproliferative neoplasms including ET (N=234) and PMF (N=50) without JAK2(V617F) or MPL exon 10 mutations. CALR mutation was detected in 53% (95% CI, 46-60%) of subjects with ET and 56% (95% CI, 41-70%) of subjects with PMF. 152 CALR mutations were identified clustering into 15 types including deletions (N=8), insertions (N=3) and complex indels (N=4). We also identified 9 new mutations. Mean (±SD) mutant allele burden was 31±12% (range, 0.5-69%). Persons with PMF had higher CALR mutant allele burdens than those with ET (38±8% vs. 29±12%; P<0.001). Amongst persons with CALR mutations, those with PMF had different clinical features from those with ET. These data may be useful for diagnosing ET and PMF in Chinese who are about 40% of all persons with ET and PMF and for monitoring therapy-response. They also highlight similarities and differences in CALR mutations between Chinese and persons of predominately European descent with these diseases.

OBJECTIVE

To analyze the relationship between six polymorphisms in genes related to oxidative stress, namely CAT-262 C>T, MnSOD Ala16Val, GPX1 Pro198Leu, GSTM1 and GSTT1 null genotypes, and GSTP1 Ile105Val, and the occurrence of BCR-ABL negative myeloproliferative neoplasms (polycythemia vera, essential thrombocythemia, and primary myelofibrosis).

METHODS

We genotyped for these polymorphisms 328 patients with a known mutation status for JAK2 V617F, MPL and CALR, and 363 controls, using molecular genetics assays.

RESULTS

The CAT-262 C>T and GPX1 Pro198Leu polymorphisms were seen significantly less frequently, while the GSTP1 IleVal105 polymorphism was seen significantly more frequently in patients with BCR-ABL negative myeloproliferative neoplasms, regardless of the molecular sub-type (e.g. JAK2 V617F or CALR mutated).

CONCLUSIONS

Our study provides evidence that variation in genes related to oxidative stress might modulate the risk of developing BCR-ABL negative myeloproliferative neoplasms.

The myeloproliferative neoplasms (MPN) are clonal, hematological malignancies that include polycythemia vera, essential thrombocythemia and primary myelofibrosis. While most cases of MPN are sporadic in nature, a familial pattern of inheritance is well recognised. The phenotype and status of the commonly acquired JAK2 V617F, CALR exon 9 and MPL W515L/K mutations in affected individuals from a consecutive series of ten familial MPN (FMPN) kindred are described. Affected individuals display the classical MPN phenotypes together with one kindred identified suggestive of hereditary thrombocytosis. In affected patients the JAK2 V617F mutation is the most commonly acquired followed by CALR exon nine mutations with no MPL W515L/K mutations detected. The JAK2 V617F and CALR exon 9 mutations appear to occur at approximately the same frequency in FMPN as in the sporadic forms of these diseases. The familial nature of MPN may often be overlooked and accordingly more common than previously considered. Characterisation of these FMPN kindred may allow for the investigation of molecular events that contribute to this inheritance.

Philadelphia-negative classical myeloproliferative neoplasms (MPN) are clonal diseases characterized by driver mutations of JAK2, MPL, or CALR. Additional mutations may occur in epigenetic regulators, signaling, or splicing genes that may be useful in the prognostic assessment of MPN patients. In primary myelofibrosis, molecular-based prognostic scoring systems have been recently proposed, but few data are available to date for polycythemia vera (PV) and essential thrombocythemia (ET). In this study, we used a next generation sequencing-based 18-gene panel in 50 JAK2V617F positive PV and JAK2V617F positive ET patients from an institutional cohort investigated at diagnosis and at 3-year follow-up (3y). Disease progression at 3y was defined by a composite criterion. Patients (28 PV and 22 ET) were included according to their clinical status, with or without disease progression. At diagnosis, we found 28 additional mutations in 21 of the 50 patients. Patients with disease progression were more likely to have at least one additional mutation. There was no difference between PV and ET. All patients with two or more additional mutations exhibited disease progression at 3y. No novel mutations appeared at 3y. The allele burden increase by at least one mutation at 3y was more frequent in patients with disease progression. Our data suggest that screening for additional mutations in PV and ET could identify patients at a higher risk of disease progression. © 2017 Wiley Periodicals, Inc.

The classic BCR-ABL1-negative myeloproliferative neoplasm is an operational sub-category of MPNs that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2V617F mutation is found in ~ 95% of PV and 50-60% of ET or PMF. In most of the remaining JAK2V617F- negative PV cases, JAK2 exon 12 mutations are present. Amongst the JAK2V617F-negative ET or PMF 5-10% of patients carry mutations in the MPL gene. Prior to 2013, there was no specific molecular marker described in the remaining 30-40% ET and PMF. In December 2013, two research groups independently reported mutations in the gene CALR found specifically in ET (67-71%) and PMF (56-88%) but not in PV. Initially CALR mutations were reported mutually exclusive with JAK2 or MPL. However, co-occurrence of CALR mutations with JAK2V617F has been reported recently in a few MPN cases. Many studies have reported important diagnostic and prognostic significance of CALR mutations in ET and PMF patients and CALR mutation screening has been proposed to be incorporated into WHO diagnostic criteria for MPN. It is suggestive in diagnostic workup of MPN that CALR mutations should not be studied in MPN patients who carry JAK2 or MPL mutations. However JAK2V617F and CALR positive patients might have a different phenotype and clinical course, distinct from the JAK2-positive or CALR-positive subgroups and identification of the true frequency of these patients may be an important factor for defining the prognosis, risk factors and outcomes for MPN patients.

OBJECTIVE

To explore the mutational status of CALR, JAK2 and MPL genes in BCR-ABL negative myeloproliferative neoplasms (MPN) patients and the clinical features of MPN patients with these mutations.

METHODS

A total of 246 patients with a definite diagnosis of BCR-ABL negative MPN were enrolled from January 2009 to January 2014 into this study. Among them, there were 48 cases of polycythemia vera (PV) patients, 171 cases of essential thrombocythemia (ET) patients and 27 cases of primary myelofibrosis (PMF) patients. And CALR, JAK2 V617F, 12 exons of JAK2 and MPL W515L/K genes were amplified by PCR and sequenced directly. Clinical features were also analyzed in patients.

RESULTS

Among 246 cases of BCR-ABL-negative MPN patients, 52 cases (21.1%) had CALR mutation, 121 cases (49.2%) JAK2 V617F mutation, 0 case (0) 12 exons of JAK2 mutation, and 2 cases (0.8%) MPL W515L/K mutation, respectively. These mutations were found existing exclusively. In PV patients, the white blood cell and platelet counts in JAK2 V617F mutated group were higher than those in wild-type JAK2 V617F group, while the level of hemoglobin was higher in wild-type JAK2 V617F group (all P<0.05). In ET patients, the white blood cell count, the level of hemoglobin, the frequency of thromboembolic events and risk stratification in JAK2 V617F mutated group were higher than those in CALR mutated group (all P<0.05). In PMF patients, the level of hemoglobin in JAK2 V617F mutated group were significantly higher than those in CALR mutated group (P<0.05).

CONCLUSIONS

The proliferative level of bone marrow, risk of thromboembolic events and stratification are lower in CALR mutated patients than those in JAK2 V617F mutated patients. The pathogenic mechanism of mutated gene should be further investigated in future.

OBJECTIVE

Previously, the family of S100A proteins has been found to be associated with inflammation and myelopoiesis and to be able to induce or support myeloproliferation during chronic inflammation. Here, we studied the inflammatory myeloid-related proteins S100A4, S100A8, S100A9 and S100A12 in myeloproliferative neoplasms (MPNs) in order to assess the involvement of chronic inflammation in the pathogenesis of MPN.

METHODS

We analyzed the S100A4, S100A8, S100A9 and S100A12 mRNA and protein levels in the bone marrow and circulation of 140 patients with MPN and 15 healthy controls using Western blotting, microarray-based mRNA expression profiling and ELISA assays, respectively. In addition we performed functional studies on the proliferation-related AKT and ERK1/2 signaling pathways in MPN-derived granulocytes using Western blotting and proteomic analyses.

RESULTS

We found that the S100A mRNA levels were increased in MPN patient-derived circulatory CD34+ cells, and that their protein expression levels were also augmented in their granulocytes and bone marrow stroma cells, depending on the JAK2V617F mutation allele burden. We also found that calreticulin (CALR) mutations were related to reduced S100A8 plasma levels in primary myelofibrosis (PMF). The S100A8 plasma levels were found to be increased in MPN, the S100A9 plasma levels in PMF and essential thrombocythemia (ET), and the S100A12 plasma levels in polycythemia vera (PV). These S100A plasma levels showed a positive correlation with the systemic inflammation marker IL-8, as well as with the numbers of leukocytes and thrombocytes, depending on the JAK2V617F mutation status. Additionally, we found that heterodimeric S100A8/9 can inhibit the AKT pathway in MPN-derived granulocytes mediated by the Toll-like receptor 4 (TLR4), depending on the CALR mutation status. Conversely, we found that blocking of the receptor for advanced glycation end products (RAGE) increased the S100A8/9-mediated inhibition of AKT signaling in the MPN-derived granulocytes. Moreover, we found that heterodimeric S100A8/9 generally induced TLR4-mediated ERK1/2 dephosphorylation proportionally to the JAK2V617F mutation allele burden. TLR4/RAGE blocking prevented the S100A8/9-mediated inhibition of ERK1/2 phosphorylation in PV.

CONCLUSIONS

From our data we conclude that the S100A8 and S100A9 granulocyte and plasma levels are increased in MPN patients, along with inflammation markers, depending on their JAK2V617F mutation allele burden. We also found that S100A8/9-mediated inhibition of the proliferation-related AKT and ERK1/2 signaling pathways can be decreased by CALR mutation-dependent TLR4 blocking and increased by RAGE inhibition in MPN.

Dengue virus (DV) infection demonstrates an intriguing virus-induced intracellular membrane alteration that results in the augmentation of major histocompatibility complex (MHC) class I-restricted antigen presentation. As oppose to its biological function in attracting CD8(+) T-cells, this phenomenon appears to facilitate the immune evasion. However, the molecular events that attribute to the dysregulation of the antigen presenting mechanism (APM) by DV remain obscure. In this study, we aimed to characterize the host cell APM upon infection with all serotypes of whole DV. Cellular RNA were isolated from infected cells and the gene expressions of LMP2, LMP7, TAP1, TAP2, TAPBP, CALR, CANX, PDIA3, HLA-A and HLA-B were analyzed via quantitative PCR. The profiles of the gene expression were further validated. We showed that all four DV serotypes modulate host APM at the proteasomal level with DV2 showing the most prominent expression profile.

Myelofibrosis (MF) is a clinical manifestation of chronic BCR-ABL1-negative chronic myeloproliferative neoplasms. Splenomegaly is one of the major clinical manifestations of MF and is directly linked to splenic extramedullary hematopoiesis (EMH). EMH is associated with abnormal trafficking patterns of clonal hematopoietic cells due to the dysregulated bone marrow (BM) microenvironment leading to progressive splenomegaly. Several recent data have emphasized the role of several cytokines for splenic EMH. Alteration of CXCL12/CXCR4 pathway could also lead to splenic EMH by migrated clonal hematopoietic cells from BM to the spleen. Moreover, low Gata1 expression was found to be significantly associated with the EMH. Several gene mutations were found to be associated with significant splenomegaly in MF. In recent data, JAK2V617F homozygous mutation was associated with a larger spleen size. In other data, CALR mutations in MF were signigicantly associated with longer larger splenomegaly-free survivals than others. In addition, MF patients with ≥1 mutations in AZXL1, EZH1 or IDH1/2 had significantly low spleen reduction response in ruxolitinib treatment. Developments of JAK inhibitors, such as ruxolitinib, pacritinib, momelotinib, and febratinib enabled the effective management in MF patients. Especially, significant spleen reduction responses of the drugs were demonstrated in several randomized clinical studies, although those could not eradicate allele burdens of MF.

As a heterogeneous group of disease, myeloproliferative neoplasms (MPNs) have confused hematologists and hematopathologists with their protean clinical presentations and myriads of morphologies. A thought of classifying MPNs based on molecular alterations has gained popularity because there is increasing evidence that molecular or chromosomal alterations have a better correlation with clinical presentation, response to therapies, and prognosis than conventional morphological classification. This type of efforts has been facilitated by the advancement of molecular technologies. A significant number of gene mutations have been identified in MPNs with JAK2 and MPL being the major ones. However, a significant gap is present in that many cases of MPNs do not harbor any of these mutations. This gap is recently filled by the discovery of Calreticulin (CALR) mutation in MPNs without JAK2 or MPL mutation and since then, the clinical and molecular correlation in MPNs has become a hot research topic. There seems to be a fairly consistent correlation between CALR mutation and certain hematological parameters such as a high platelet count and a better prognosis in MPNs with CALR mutation. However, controversies are present regarding the risks of thrombosis, interactions of CALR with other gene mutation, the role of CALR in the pathogenesis, and the optimal treatment strategies. In addition, there are many questions remain to be answered, which all boiled down to the molecular mechanisms by which CALR causes or contributes to MPNs. Here, we summarized current published literatures on CALR mutations in MPNs with an emphasis on the clinical-molecular correlation. We also discussed the controversies and questions remain to be answered.

Janus kinase 2 (JAK2) and calreticulin (CALR) constitute the two most frequent mutations in essential thrombocythemia (ET), and both are reported to be mutually exclusive. Hence, we examined a cohort of 123 myeloproliferative neoplasm (MPN) patients without BCR-ABL1 rearrangement and additional ET patients (n=96) for coexistence of JAK2 and CALR mutations. The frequency of CALR mutations was 20.3% in 123 MPN patients; 31.1% in ET (n=74), 25% in primary myelofibrosis (n=4) and 2.2% in polycythemia vera (n=45). JAK2 and CALR mutations coexisted in 7 (4.2%) of 167 ET patients. Clinical characteristics, progression-free survival (PFS), and elapsed time to achieve partial remission across 4 groups (JAK2+/CALR+, JAK2+/CALR-, JAK2-/CALR+, JAK2-/CALR-) were reviewed. The JAK2+/CALR- group had higher leukocyte counts and hemoglobin levels and more frequent thrombotic events than JAK2-/CALR- group. JAK2 mutations have a greater effect on the disease phenotype and the clinical features of MPN patients rather than do CALR mutation. JAK2+ groups showed a tendency of poor PFS than JAK2- groups regardless of CALR mutation. CALR+ was a predictor of late response to the treatment. Our study also showed that thrombosis was more frequent in ET patients with type 2 CALR mutations than in those with type 1 CALR mutations.