Increased Dkk‐1 plasma levels may discriminate disease subtypes in myeloproliferative neoplasms

Alterations in the bone marrow niche induced by abnormal production of cytokines and other soluble factors have been associated with disease progression in classical BCR‐ABL1 negative myeloproliferative neoplasms (MPN). Variations in circulating proteins might reflect local disease processes and plasma proteome profiling could serve to identify possible diagnostic and prognostic biomarkers. We employed a human cytokine array to screen for 105 distinct analytes in pooled plasma samples obtained from untreated young MPN patients (<35 years) with different clinical phenotypes and driver mutations, as well as from healthy individuals. Among molecules that exhibited significantly increased levels in MPN patients versus controls, the top of the list was represented by Dickkopf‐related protein 1 (Dkk‐1), which also showed the highest potential for discrimination between MPN subtypes. In the next step, a quantitative ELISA was used to measure plasma Dkk‐1 levels in 30 young‐onset MPN—10 essential thrombocythemia (ET), 10 polycythemia vera (PV), 10 pre‐fibrotic primary myelofibrosis (pre‐PMF)—and 10 controls. The results suggested that plasma Dkk‐1 levels could differentiate ET from pre‐PMF, in JAK2 V617F‐positive as well as in CALR‐positive patients, and also ET from PV in JAK2 V617F‐positive patients.     

Microarray and Proteomic Analyses of Myeloproliferative Neoplasms with a Highlight on the mTOR Signaling Pathway

The gene and protein expression profiles in myeloproliferative neoplasms (MPNs) may reveal gene and protein markers of a potential clinical relevance in diagnosis, treatment and prediction of response to therapy. Using cDNA microarray analysis of 25,100 unique genes, we studied the gene expression profile of CD34⁺ cells and granulocytes obtained from peripheral blood of subjects with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The microarray analyses of the CD34⁺ cells and granulocytes were performed from 20 de novo MPN subjects: JAK2 positive ET, PV, PMF subjects, and JAK2 negative ET/PMF subjects. The granulocytes for proteomic studies were pooled in 4 groups: PV with JAK2 mutant allele burden above 80%, ET with JAK2 mutation, PMF with JAK2 mutation and ET/PMF with no JAK2 mutation. The number of differentially regulated genes was about two fold larger in CD34⁺ cells compared to granulocytes. Thirty-six genes (including RUNX1, TNFRSF19) were persistently highly expressed, while 42 genes (including FOXD4, PDE4A) were underexpressed both in CD34⁺ cells and granulocytes. Using proteomic studies, significant up-regulation was observed for MAPK and PI3K/AKT signaling regulators that control myeloid cell apoptosis and proliferation: RAC2, MNDA, S100A8/9, CORO1A, and GNAI2. When the status of the mTOR signaling pathway related genes was analyzed, PI3K/AKT regulators were preferentially up-regulated in CD34⁺ cells of MPNs, with down-regulated major components of the protein complex EIF4F. Molecular profiling of CD34⁺ cells and granulocytes of MPN determined gene expression patterns beyond their recognized function in disease pathogenesis that included dominant up-regulation of PI3K/AKT signaling.     

Splenomegaly in myelofibrosis-new options for therapy and the therapeutic potential of Janus kinase 2 inhibitors

ABSTRACT: Splenomegaly is a common sign of primary myelofibrosis (PMF), post-polycythemia vera myelofibrosis (post-PV MF), and post-essential thrombocythemia myelofibrosis (post-ET MF) that is associated with bothersome symptoms, which have a significant negative impact on patients' quality of life. It may also be present in patients with advanced polycythemia vera (PV) or essential thrombocythemia (ET). Until recently, none of the therapies used to treat MF were particularly effective in reducing splenomegaly. The discovery of an activating Janus kinase 2 (JAK2) activating mutation (JAK2V617F) that is present in almost all patients with PV and in about 50-60?% of patients with ET and PMF led to the initiation of several trials investigating the clinical effectiveness of various JAK2 (or JAK1/JAK2) inhibitors for the treatment of patients with ET, PV, and MF. Some of these trials have documented significant clinical benefit of JAK inhibitors, particularly in terms of regression of splenomegaly. In November 2011, the US Food and Drug Administration approved the use of the JAK1- and JAK2-selective inhibitor ruxolitinib for the treatment of patients with intermediate or high-risk myelofibrosis, including PMF, post-PV MF, and post-ET MF. This review discusses current therapeutic options for splenomegaly associated with primary or secondary MF and the treatment potential of the JAK inhibitors in this setting.     

Co‐targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms

Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation‐specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single‐agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild‐type counterpart, and preferentially prevented colony formation from JAK2V617F knock‐in mice and patients' progenitor cells compared with normal ones. Co‐treatment of BEZ235 and ruxolitinib produced significant synergism in all these in‐vitro models. Co‐treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F‐mutated Ba/F3‐EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock‐in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN.     

In vitro megakaryocyte differentiation and proplatelet formation in Ph-negative classical myeloproliferative neoplasms: distinct patterns in the different clinical phenotypes

Background

Ph-negative myeloproliferative neoplasms (MPNs) are clonal disorders that include primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocythemia (ET). Although the pathogenesis of MPNs is still incompletely understood, an involvement of the megakaryocyte lineage is a distinctive feature.

Methodology/Principal Findings

We analyzed the in vitro megakaryocyte differentiation and proplatelet formation in 30 PMF, 8 ET, 8 PV patients, and 17 healthy controls (CTRL). Megakaryocytes were differentiated from peripheral blood CD34⁺ or CD45⁺ cells in the presence of thrombopoietin. Megakaryocyte output was higher in MPN patients than in CTRL with no correlation with the JAK2 V617F mutation. PMF-derived megakaryocytes displayed nuclei with a bulbous appearance, were smaller than ET- or PV-derived megakaryocytes and formed proplatelets that presented several structural alterations. In contrast, ET- and PV-derived megakaryocytes produced more proplatelets with a striking increase in bifurcations and tips compared to both control and PMF. Proplatelets formation was correlated with platelet counts in patient peripheral blood. Patients with pre-fibrotic PMF had a pattern of megakaryocyte proliferation and proplatelet formation that was similar to that of fibrotic PMF and different from that of ET.

Conclusions/Significance

In conclusion, MPNs are associated with high megakaryocyte proliferative potential. Profound differences in megakaryocyte morphology and proplatelet formation distinguish PMF, both fibrotic and prefibrotic, from ET and PV.     

Recognizing and managing the expanded risk of tumor lysis syndrome in hematologic and solid malignancies

Background

Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34⁺ hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters.

Results

By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34⁺ cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34⁺ cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-X _L and BCLW. In contrast, pro-apoptotic BID and BIM _EL expression were downregulated in ET leukocytes. Increased BCL-X_L protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly.

Conclusions

Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.     

The Polymorphisms in LNK Gene Correlated to the Clinical Type of Myeloproliferative Neoplasms

ObjectiveLNK is an adapter protein negatively regulating the JAK/STAT cell signaling pathway. In this study, we observed the correlation between variation in LNK gene and the clinical type of myeloproliferative neoplasms (MPN).MethodsA total of 285 MPN cases were recruited, including essential thrombocythemia (ET) 154 cases, polycythemia vera (PV) 76 cases, primary myelofibrosis (PMF) 19 cases, and chronic myeloid leukemia (CML) 36 cases. Ninety-three healthy individuals were used as normal controls. V617F mutation in JAK2 was identified by allele-specific PCR method, RT-PCR was used for the detection of BCR/ABL1 fusion gene, and mutations and variations in coding exons and their flanking sequences of LNK gene were examined by PCR-sequencing.ResultsMissense mutations of A300V, V402M, and R415H in LNK were found in 8 patients including ET (4 cases, all combined with JAK2-V617F mutation), PV (2 cases, one combined with JAK2-V617F mutation), PMF (one case, combined with JAK2-V617F mutation) and CML (one case, combined with BCR/ABL1 fusion gene). The genotype and allele frequencies of the three SNPs (rs3184504, rs111340708 and rs78894077) in LNK were significantly different between MPN patients and controls. For rs3184504 (T/C, in exon2), the T allele (p.262W) and TT genotype were frequently seen in ET, PV and PMF (P<0.01), and C allele (p.262R) and CC genotype were frequently seen in CML (P<0.01). For rs78894077 (T/C, in exon1), the T allele (p.242S) was frequently found in ET (P<0.05). For rs111340708 (TGGGGx5/TGGGGx4, in intron 5), the TGGGG x4 allele was infrequently found in ET, PMF and CML(P<0.01).ConclusionMutations in LNK could be found in some of MPN patients in the presence or absence of JAK2-V617F mutation. Several polymorphisms in LNK gene may affect the clinical type or the genetic predisposition of MPN.     

A novel BH3 mimetic efficiently induces apoptosis in melanoma cells through direct binding to anti‐apoptotic Bcl‐2 family proteins,including phosphorylated Mcl‐1

The Bcl‐2 family modulates sensitivity to chemotherapy in many cancers, including melanoma, in which the RAS/BRAF/MEK/ERK pathway is constitutively activated. Mcl‐1, a major anti‐apoptotic protein in the Bcl‐2 family, is extensively expressed in melanoma and contributes to melanoma's well‐documented chemoresistance. Here, we provide the first evidence that Mcl‐1 phosphorylation at T163 by ERK1/2 and JNK is associated with the resistance of melanoma cell lines to the existing BH3 mimetics gossypol, S1 and ABT‐737, and a novel anti‐apoptotic mechanism of phosphorylated Mcl‐1 (pMcl‐1) is revealed. pMcl‐1 antagonized the known BH3 mimetics by sequestering pro‐apoptotic proteins that were released from Bcl‐2/Mcl‐1. Furthermore, an anthraquinone BH3 mimetic, compound 6, was identified to be the first small molecule to that induces endogenous apoptosis in melanoma cells by directly binding Bcl‐2, Mcl‐1, and pMcl‐1 and disrupting the heterodimers of these proteins. Although compound 6 induced upregulation of the pro‐apoptotic protein Noxa, its apoptotic induction was independent of Noxa. These data reveal the promising therapeutic potential of targeting pMcl‐1 to treat melanoma. Compound 6 is therefore a potent drug that targets pMcl‐1 in melanoma.     

Activated apoptotic and anti‐survival effects on rat hearts with fructose induced metabolic syndrome

Consumption of fructose has been linked to the development of metabolic syndrome, whereas the cardiomyopathic changes and cardiac apoptosis of dietary high‐fructose intake have not yet been clarified. The purpose of this study was to evaluate the effects of high‐fructose on cardiac apoptotic and survival pathways. Thirty‐two Wistar rats were randomly divided into a control group (CON), which received a standard chow diet, and a fructose‐induced metabolic syndrome group (FIMS), which received a 50% fructose‐content diet for 13 weeks. Histopathological analysis, TUNEL assays and Western blotting were performed on the excised hearts from both groups. The blood pressure, glucose, insulin, triglyceride and cholesterol levels were significantly increased in the FIMS group, compared with the CON group. The abnormal myocardial architecture, enlarged interstitial space and increased cardiac TUNEL‐positive apoptotic cells were observed in the FIMS group. The TNF‐α, TNF receptor 1, Fas ligand, Fas receptor, FADD, and activated caspase‐3 and 8 protein levels (Fas pathway) and the Bax, Bak, Bax/Bcl‐2, Bak/Bcl‐xL, cytosolic cytochrome c, and activated caspase‐3 and nine protein levels (mitochondria pathway) were increased in the FIMS group compared with those in the CON group. The IGFI, IGFI‐R, p‐PI3K, p‐Akt, Bcl‐2 and Bcl‐xL protein levels (survival pathway) were all significantly decreased in the FIMS group compared with those in the CON group. High‐fructose intake elevated blood pressure and glucose levels; moreover, high‐fructose diet activated cardiac Fas‐dependent and mitochondria‐dependent apoptotic pathways and suppressed the survival pathway, which might provide one possible mechanism for developing heart failure in patients with metabolic syndrome. Copyright © 2013 John Wiley & Sons, Ltd.     

Role of p38 MAPK in enhanced human cancer cells killing by the combination of aspirin and ABT‐737

Regular use of aspirin after diagnosis is associated with longer survival among patients with mutated‐PIK3CA colorectal cancer, but not among patients with wild‐type PIK3CA cancer. In this study, we showed that clinically achievable concentrations of aspirin and ABT‐737 in combination could induce a synergistic growth arrest in several human PIK3CA wild‐type cancer cells. In addition, our results also demonstrated that long‐term combination treatment with aspirin and ABT‐737 could synergistically induce apoptosis both in A549 and H1299 cells. In the meanwhile, short‐term aspirin plus ABT‐737 combination treatment induced a greater autophagic response than did either drug alone and the combination‐induced autophagy switched from a cytoprotective signal to a death‐promoting signal. Furthermore, we showed that p38 acted as a switch between two different types of cell death (autophagy and apoptosis) induced by aspirin plus ABT‐737. Moreover, the increased anti‐cancer efficacy of aspirin combined with ABT‐737 was further validated in a human lung cancer A549 xenograft model. We hope that this synergy may contribute to failure of aspirin cancer therapy and ultimately lead to efficacious regimens for cancer therapy.     

JAK2 inhibition has different therapeutic effects according to myeloproliferative neoplasm development in mice

JAK2 inhibition therapy is used to treat patients suffering from myeloproliferative neoplasms (MPN). Conflicting data on this therapy are reported possibly linked to the types of inhibitors or disease type. Therefore, we decided to compare in mice the effect of a JAK2 inhibitor, Fedratinib, in MPN models of increasing severity: polycythemia vera (PV), post‐PV myelofibrosis (PPMF) and rapid post‐essential thrombocythemia MF (PTMF). The models were generated through JAK2 activation by the JAK2^V617F mutation or MPL constant stimulation. JAK2 inhibition induced a correction of splenomegaly, leucocytosis and microcytosis in all three MPN models. However, the effects on fibrosis, osteosclerosis, granulocytosis, erythropoiesis or platelet counts varied according to the disease severity stage. Strikingly, complete blockade of fibrosis and osteosclerosis was observed in the PPMF model, linked to correction of MK hyper/dysplasia, but not in the PTMF model, suggesting that MF development may also become JAK2‐independent. Interestingly, we originally found a decreased in the JAK2^V617F allele burden in progenitor cells from the spleen but not in other cell types. Overall, this study shows that JAK2 inhibition has different effects according to disease phenotypes and can (i) normalize platelet counts, (ii) prevent the development of marrow fibrosis/osteosclerosis at an early stage and (iii) reduce splenomegaly through blockage of stem cell mobilization in the spleen.     

Sex differences in the level of Bcl‐2 family proteins and caspase‐3 activation in the sexually dimorphic nuclei of the preoptic area in postnatal rats

In developing rats, sex differences in the number of apoptotic cells are found in the central division of the medial preoptic nucleus (MPNc), which is a significant component of the sexually dimorphic nucleus of the preoptic area, and in the anteroventral periventricular nucleus (AVPV). Specifically, male rats have more apoptotic cells in the developing AVPV, whereas females have more apoptotic cells in the developing MPNc. To determine the mechanisms for the sex differences in apoptosis in these nuclei, we compared the expression of the Bcl‐2 family members and active caspase‐3 in postnatal female and male rats. Western blot analyses for the Bcl‐2 family proteins were performed using preoptic tissues isolated from the brain on postnatal day (PD) 1 (day of birth) or on PD8. In the AVPV‐containing tissues of PD1 rats, there were significant sex differences in the level of Bcl‐2 (female > male) and Bax (female < male) proteins, but not of Bcl‐xL or Bad proteins. In the MPNc‐containing tissues of PD8 rats, there were significant sex differences in the protein levels for Bcl‐2 (female < male), Bax (female > male), and Bad (female < male), but not for Bcl‐xL. Immunohistochemical analyses showed significant sex differences in the number of active caspase‐3‐immunoreactive cells in the AVPV on PD1 (female < male) and in the MPNc on PD8 (female > male). We further found that active caspase‐3‐immunoreactive cells of the AVPV and MPNc were immunoreactive for NeuN, a neuronal marker. These results suggest that there are sex differences in the induction of apoptosis via the mitochondrial pathway during development of the AVPV and MPNc. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

LIGHT/IFN‐γ triggers β cells apoptosis via NF‐κB/Bcl2‐dependent mitochondrial pathway

LIGHT recruits and activates naive T cells in the islets at the onset of diabetes. IFN‐γ secreted by activated T lymphocytes is involved in beta cell apoptosis. However, whether LIGHT sensitizes IFNγ‐induced beta cells destruction remains unclear. In this study, we used the murine beta cell line MIN6 and primary islet cells as models for investigating the underlying cellular mechanisms involved in LIGHT/IFNγ – induced pancreatic beta cell destruction. LIGHT and IFN‐γ synergistically reduced MIN6 and primary islet cells viability; decreased cell viability was due to apoptosis, as demonstrated by a significant increase in Annexin V⁺ cell percentage, detected by flow cytometry. In addition to marked increases in cytochrome c release and NF‐κB activation, the combination of LIGHT and IFN‐γ caused an obvious decrease in expression of the anti‐apoptotic proteins Bcl‐2 and Bcl‐xL, but an increase in expression of the pro‐apoptotic proteins Bak and Bax in MIN6 cells. Accordingly, LIGHT deficiency led to a decrease in NF‐κB activation and Bak expression, and peri‐insulitis in non‐obese diabetes mice. Inhibition of NF‐κB activation with the specific NF‐κB inhibitor, PDTC (pyrrolidine dithiocarbamate), reversed Bcl‐xL down‐regulation and Bax up‐regulation, and led to a significant increase in LIGHT‐ and IFN‐γ‐treated cell viability. Moreover, cleaved caspase‐9, ‐3, and PARP (poly (ADP‐ribose) polymerase) were observed after LIGHT and IFN‐γ treatment. Pretreatment with caspase inhibitors remarkably attenuated LIGHT‐ and IFNγ‐induced cell apoptosis. Taken together, our results indicate that LIGHT signalling pathway combined with IFN‐γ induces beta cells apoptosis via an NF‐κB/Bcl2‐dependent mitochondrial pathway.     

Calcium‐stimulated mitogen‐activated protein kinase activation elicits Bcl‐xL downregulation and Bak upregulation in notexin‐treated human neuroblastoma SK‐N‐SH cells

Notechis scutatus scutatus notexin induced apoptotic death of SK‐N‐SH cells accompanied with downregulation of Bcl‐xL, upregulation of Bak, mitochondrial depolarization, and ROS generation. Upon exposure to notexin, Ca²⁺‐mediated JNK and p38 MAPK activation were observed in SK‐N‐SH cells. Production of ROS was a downstream event followed by Ca²⁺‐mediated mitochondrial alteration. Notexin‐induced cell death, mitochondrial depolarization, and ROS generation were suppressed by SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). Moreover, phospho‐p38 MAPK and phospho‐JNK were proved to be involved in Bcl‐xL degradation, and overexpression of Bcl‐xL attenuated the cytotoxic effect of notexin. Bak upregulation was elicited by p38 MAPK‐mediated ATF‐2 activation and JNK‐mediated c‐Jun activation. Suppression of Bak upregulation by ATF‐2 siRNA or c‐Jun siRNA attenuated notexin‐evoked mitochondrial depolarization and rescued viability of notexin‐treated cells. Taken together, our data indicate that notexin‐induced apoptotic death of SK‐N‐SH cells is mediated through mitochondrial alteration triggering by Ca²⁺‐evoked p38 MAPK/ATF‐2 and JNK/c‐Jun signaling pathways. J. Cell. Physiol. 222:177–186, 2010. © 2009 Wiley‐Liss, Inc.     

IBRDC2, an IBR‐type E3 ubiquitin ligase,is a regulatory factor for Bax and apoptosis activation

Bax, a pro‐apoptotic protein from the Bcl‐2 family, is central to apoptosis regulation. To suppress spontaneous apoptosis, Bax must be under stringent control that may include regulation of Bax conformation and expression levels. We report that IBRDC2, an IBR‐type RING‐finger E3 ubiquitin ligase, regulates the levels of Bax and protects cells from unprompted Bax activation and cell death. Downregulation of IBRDC2 induces increased cellular levels and accumulation of the active form of Bax. The ubiquitination‐dependent regulation of Bax stability is suppressed by IBRDC2 downregulation and stimulated by IBRDC2 overexpression in both healthy and apoptotic cells. Although mostly cytosolic in healthy cells, upon induction of apoptosis, IBRDC2 accumulates in mitochondrial domains enriched with Bax. Mitochondrial accumulation of IBRDC2 occurs in parallel with Bax activation and also depends on the expression levels of Bcl‐xL. Furthermore, IBRDC2 physically interacts with activated Bax. By applying Bax mutants in HCT116 Bax^?/? cells, combined with the use of active Bax‐specific antibodies, we have established that both mitochondrial localization and apoptotic activation of Bax are required for IBRDC2 translocation to the mitochondria.