Overproduction Of Cells Research Articles

In vitro and in vivo effects of JAK2 inhibition in chronic myelomonocytic leukemia.

In chronic myelomonocytic leukemia (CMML), colony-forming units granulocyte/macrophage (CFU-GM), which grow in vitro in the absence of exogenous growth factors, arise from the abnormal clone that is responsible for the overproduction of granulomonocytic cells. Previous in vitro findings including ours suggest that divergent molecular aberrations in CMML seem to converge within the GM-CSF signaling pathway. As JAK2 is a sentinel kinase in this pathway, JAK2 inhibition may be an attractive treatment approach in CMML. We investigated the in vitro effects of the specific JAK2 inhibitor TG101209 on the autonomous CFU-GM formation from peripheral blood mononuclear cells of patients with CMML. TG101209 was found to either block or strongly inhibit spontaneous CFU-GM growth in all 10 patients tested. This inhibitory effect was dose dependent and significantly more pronounced as compared to the inhibitory effect on stimulated CFU-GM growth from normal individuals. In a CMML patient with splenomegaly, who was treated with the JAK1/2 inhibitor ruxolitinib off label, we can demonstrate a spleen response and the disappearance of constitutional symptoms which was associated with a decrease in autonomous CFU-GM formation ex vivo. Pharmacological JAK2 inhibition may be an interesting approach to be systematically studied in patients with CMML.

Modulation of neutrophil gelatinase-associated lipocalin2 (NGAL, oncogene 24/p3), proinflammatory cytokines (PIC), cystatin C and lipid rafts disruption (LR) with HMG-CoA reductase inhibitor simvastatin (SV) in patients with JAK2V617F myeloprliferative neoplasms (MPN).

e18560Background: MPN is a manifestation of diseases with overproduction of myoerythroid cells, platelets, BCR/abl fusion genes with prevalence to polycythemia Vera (PCV), essential thrombocythemia...

In This Issue

HomeCirculation ResearchVol. 118, No. 7In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Originally published1 Apr 2016https://doi.org/10.1161/RES.0000000000000102Circulation Research. 2016;118:1041Glut1, Myelopoiesis, and Atherosclerosis (p 1062)Download figureDownload PowerPointInhibiting extramedullary hematopoietic activity may halt progression of atherosclerosis, say Sarrazy et al.To proliferate and differentiate hematopoietic stem and progenitor cells (HSPCs) in the bone marrow increase their glucose uptake to meet the rising metabolic demand. Indeed PET-CT imaging can identify increased metabolic activity in HSPCs by the uptake of glucose (or rather its radiolabeled analogue) during bone marrow and extramedullary hematopoietic activity. Cell proliferation dependent glucose uptake has been observed in atherosclerotic plaques as well as the spleen. Overproduction of hematopoietic cells, including neutrophils and monocytes, could both contribute to atherosclerosis onset and exacerbate plaque growth. To study extramedullary hematopoiesis more closely, Sarrazy and colleagues examined atherosclerosis-prone mice. They found that glucose uptake was increased in the aortic arch, spleens and bone marrow of these animals, which correlated with increased expression of the glucose transporter Glut1 in their HSPCs. Furthermore, suppression of Glut1 activity reduced not only HSPC proliferation and differentiation—specifically myelopoiesis—but also the progression of atherosclerosis. The authors propose that preventing glucose uptake by HSPCs with an appropriately targeted Glut1 inhibitor could be a potential treatment strategy for atherosclerosis.Long-Term Effects of Cardiac Progenitor Cells (p 1091)Download figureDownload PowerPointThe benefits of cardiac progenitor cell therapy lasts at least a year in rats, report Tang et al.Several studies show that the therapeutic use of cardiac progenitor cells (CPCs) can promote repair and improve function of damaged hearts in animals after myocardial infarction. However, long-term effects of CPC therapy remain unknown as most studies have examined the potential benefits for only four-to-six weeks. Therefore, Tang and colleagues studied the outcome of CPC therapy for myocardial infarction in rats, one year after CPC transplant. They found that while the overall survival rate was no different between control and CPC-treated animals, the treated group exhibited less hypertrophy, smaller scars, increased left ventricle wall thickness, and evidence of less fibrosis. Significantly, CPC treatment did not result in tumor formation. The team used male CPCs and female recipients to enable identification of the cells—by the Y chromosome—and this approach revealed that while a small number of the cells were still present even after 1 year, the transplanted cells had not differentiated into mature cardiomyocytes, although increased proliferation of endogenous CPCs was observed. Together these results indicate that CPC therapy is safe, and that it provides long-term benefits, and induces endogenous CPCs to proliferate and promote repair. The study paves the way for long-term studies evaluating the clinical efficacy of stem cells, say the authors.GRK2 and Prognosis in Heart Failure (p 1116)Download figureDownload PowerPointRengo et al assess the usefulness of a possible new biomarker for heart failure.Heart failure (HF) is a leading cause of death worldwide, and about five million people in the United States suffer from the condition. A major characteristic of HF is chronic sympathetic nervous system (SNS) hyperactivity, which while compensatory in the initial stages of disease becomes deleterious in the long run. Indeed, chronic SNS hyperactivity boosts cardiac levels of G-protein coupled receptor (GPCR) kinase 2 (GRK2), which in turn leads to dysfunctional β-adrenergic signaling—a key factor contributing to the progress of HF pathology. Importantly, the levels of GRK2 in peripheral lymphocytes correlate with those in the failing heart. Rengo and colleagues studied levels of lymphocyte GRK2 in 257 HF patients and found that they accurately reflect disease severity. Patients at low risk of death has lower levels of GRK2, while high levels of protein were identified in high risk individuals. Currently, left ventricle ejection fraction and levels of NT-proBNP are used as biomarkers for HF; however, the team suggests that GRK2 could offer independent and additional prognostic value over and above current approaches. The authors therefore suggest that further larger-scale clinical investigations are warranted to assess the value of the prognostic value of GRK2 in heart failure patients. Previous Back to top Next FiguresReferencesRelatedDetails April 1, 2016Vol 118, Issue 7 Advertisement Article InformationMetrics © 2016 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000102 Originally publishedApril 1, 2016 PDF download Advertisement

Contemporary approach to essential thrombocythemia and polycythemia vera.

Management of polycythemia vera and essential thrombocythemia requires understanding of the key concepts regarding diagnosis, risk stratification, and management. Essential thrombocythemia and polycythemia vera are among the Philadelphia chromosome negative myeloproliferative neoplasms. They are characterized by overproduction of blood cells and their complications include thrombosis, hemorrhage, and progression to myelofibrosis or acute myeloid leukemia (AML). Management of essential thrombocythemia/polycythemia vera requires recognition of the risk factors for thrombosis and hemorrhage. Risk stratification allows the clinician to make a treatment plan that may include antiplatelet therapy with aspirin alone or in combination with therapeutic phlebotomy in the case of polycythemia vera, or cytoreductive therapy for high-risk patients with either essential thrombocythemia or polycythemia vera. Hydroxyurea remains first-line therapy for high-risk patients with essential thrombocythemia/polycythemia vera, whereas second-line options include anagrelide, pegylated-IFNα-2a, and the JAK1/2 inhibitor ruxolitinib. The current evaluation of pegylated-IFNα-2a in global phase II and III studies will provide clarity to the potential long-term benefit and risks associated with this biologic in patients with essential thrombocythemia/polycythemia vera. Novel therapeutics aimed at prevention of disease progression to myelofibrosis/AML are the focus of current clinical trials. Risk stratification of patients with essential thrombocythemia/polycythemia vera by age and/or history of thrombosis provides the basis of risk adapted therapeutic intervention. Aggressive control of modifiable cardiovascular risk factors, the use of antiplatelet agents, control of the hematocrit less than 45% in polycythemia vera, and cytoreductive therapy in high-risk essential thrombocythemia/polycythemia vera patients is the focus of management. The exact role of IFN-α remains undefined and under active investigation, and the recent approval of ruxolitinib provides patients with polycythemia vera a second-line option.

Protein homeostasis limiting cancer

A protein involved in protein homeostasis limits the maturation of IGF1R and the overproduction of blood cells.

Nod-like receptor protein 3 inflammasome activation by Escherichia coli RNA induces transforming growth factor beta 1 secretion in hepatic stellate cells.

Nod-like receptor protein 3 (NLRP3) inflammasome has been implicated in alcoholic liver disease. Chronic alcohol consumption enhances gut permeability and causes microbial translocation. The present study explored the activation of the NLRP3 inflammasome by Escherichia coli RNA in hepatic stellate cells (HSCs), and the potential role of NLRP3 inflammasome in hepatic fibrosis. E. coli RNA transfection induced HSC-T6 cells to secrete and express mature interleukin-1 beta (IL-1β), which was abolished by NLRP3 siRNA pretreatment. In addition, E. coli RNA transfection enhanced caspase-1 expression, whereas reduced caspase-1 precursor (pro-caspase-1) expression. E. coli RNA-stimulated transforming growth factor beta 1 (TGF-β1) overproduction in HSC-T6 cells, which was blocked by recombinant IL-1 receptor antagonist (rIL-1Ra) or nuclear factor κB inhibitor BAY 11-7082. Furthermore, E. coli RNA-induced overexpression of pro-fibrogenic factors was suppressed by rIL-1Ra or TGF-β receptor inhibitor A83-01. These results demonstrate that E. coli RNA can stimulate NLRP3 inflammasome activation, which leads to excessive production of pro-fibrogenic factors, suggesting that NLRP3 inflammasome activation in HSCs may play a role in hepatic fibrosis.

Topical Application of Cudrania tricuspidata Stem Extract Inhibits Atopic Dermatitis-Like Skin Lesions in an NC/Nga Mouse Model: An Experimental Animal Study

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by elevated immunoglobulin E (IgE), mast cell infiltration and skin lesions including pruritus, erythema and eczema. Cudrania tricuspidata extracts have been clinically administered for a long time in the East Asia including Korean and China as a home-remedy to diminish the inflammation of gastritis and hepatitis. To examine whether it works on AD or not, an AD-like animal model was experimented in this study. AD was induced by applying Dermatophagoides farinae (D. farinae) extract to the backs of 9-week old NC/Nga mice for 21 days. Following this, an ethanol extract of C. tricuspidata stems (EECT) was applied topically for 14 days to the sensitized skin, while distilled water was used as a control (EECT0 mice). Anti-AD effects of EECT were evaluated using scores for AD-like skin lesions, serum IgE levels and mast cell counts in the skin dermal layers to assess inflammation. Topically applied ethanol extract of Cudrania tricuspidata stems (EECT 7.5, 25 and 75 mg/mL) markedly reduced AD-like skin lesions after 4 days (by 30.1%, 31.4% and 38.5%, respectively) and also after 14 days (by 63.6%, 66.1% and 49.6%, respectively), while distilled water improved AD by 17.8% and 38.7%, respectively (p D. farinae extract (p = 0.003) and EECT attenuated the mast cell overproduction, and reduced mast cell degranulation markedly. Attenuation was most obvious in the early stage of EECT treatment when the AD was most acute.

JAK2V617F-Mutant Megakaryocytes Contribute to Stem Cell Expansion in Myeloproliferative Neoplasms

IntroductionThe myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET), are characterized by unregulated hematopoietic stem cell (HSC) expansion and overproduction of blood cells. The JAK2V617F mutation can be found in majority of MPN patients, but the mechanisms responsible for the MPN stem cell expansion remain incomplete. One hallmark feature of MPNs is megakaryocyte (MK) hyperplasia. We hypothesize that JAK2V617F -mutant MKs contribute to MPN pathogenesis by promoting HSC expansion.MethodsJAK2V617F Flip-Flop (FF1) mice and Pf4-Cre mice (from Radek Skoda, Switzerland) were crossed to generate a MK cell lineage-specific human JAK2V617F knock-in mouse strain (Pf4/FF1). Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline. Mice were sacrificed at 28 wk of age. Bone marrow and spleen cell flow cytometry analysis was performed on a FACSAriaTM III (BD). Mouse methylcellulose media and Megacult-C media (Stem Cell Technologies) were used to assay colony formation. The TaqMan¨ Gene Expression Assay (Applied Biosystems) was used for quantitative real-time polymerase chain reaction (qRT-PCR) to verify differential expression of fibroblast growth factor 1 (FGF1), platelet factor 4 (Pf4), transforming growth factor beta 1 (TGFb-1), chemokine (C-X-C motif) ligand 12 (CXCL12). For HSC transplantation experiments, wild-type CD45.1+ recipient mice (B6.SJL, Taconic) were lethally irradiated (950 cGy) and then received 5 x 106 nucleated marrow cells from Pf4/FF1 or age-matched littermate control mice (CD45.2).ResultsThrombocytosis was observed in Pf4/FF1 mice at 16wk of age. At 28wk there was a significant increase in platelet count (1406 vs. 808 x 109/L, p = 0.0009) in Pf4/FF1 mice but no change was noted in hemoglobin or white blood cell count compared with age-matched littermate controls. Splenomegaly was observed in 28wk old Pf4/FF1 mice compared to controls (spleen weight 0.101 vs. 0.070 gram, p = 0.040). Marrow cell colony assays demonstrated an increase in total hematopoietic progenitors (1.8-fold, p = 0.007) and in MK-committed progenitors (1.9-fold, p = 0.00009) in Pf4/FF1 mice compared to controls (Figure 1). Marrow cell flow cytometry analysis revealed that CD45+ EPCR+ CD48- CD150+ (E-SLAM) cells, a highly purified HSC population, were increased 3.2-fold (p = 0.028, n=4) in Pf4/FF1 mice compared to control mice, although the cells did not express JAK2V617F as determined by RT-PCR for FF1. CD41+ MK cells were also increased in Pf4/FF1 mice (Figure 2). Histologic analysis of marrow sections revealed an increased megakaryopoiesis with more MKs adjacent to the endosteal surface in Pf4/FF1 mice than in controls. In addition, there were dilated marrow sinusoids in Pf4/FF1 mice and sinusoidal endothelial cells (EC) were surrounded by MKs that conformed to the EC morphology (Figure 3). No marrow or spleen fibrosis was observed at 28 wk of age. To more directly test the effects of JAK2V617F -mutant MKs on HSCs, marrow cells from 28-wk old Pf4/FF1 mice or controls (CD45.2) were transplanted into lethally irradiated congenic CD45.1 recipients (n = 5 in each group). By 12 wks post transplantation, recipients of Pf4/FF1 marrow had already developed a higher platelet count (1105 vs. 772 x 109/L, p = 0.025) and a mildly elevated red cell count (10.7 vs. 9.9 x 1012/L, p = 0.017) than mice transplanted with the control marrow. To begin to understand the MK signals responsible for HSC expansion in Pf4/FF1 mice, qRT-PCR of purified CD41+ MK cells identified up-regulation of FGF1 (8.1-fold, p = 0.02), Pf4 (8.0-fold, p = 0.0003), and CXCL12 (3.1-fold, p = 0.005) and down-regulation of TGFβ-1 (75%, p = 0.0005) in Pf4/FF1 mice compared to controls.ConclusionsPf4/FF1 mice developed an ET phenotype with thrombocytosis, splenomegaly and increased numbers of E-SLAM HSCs and CD41+ MK cells, despite JAK2V617F expression being restricted to MKs. Marrow histology suggested that both the osteoblastic niche and vascular niche might be affected by the JAK2V617F -mutant MKs. In addition, marrow cells from Pf4/FF1 mice repopulated wild-type recipients and induced a MPN phenotype. MKs from such mice displayed an altered pattern of cytokine expression, possibly contributing to the observed effects but the precise mechanism by which JAK2V617F-bearing MKs affect the MPN marrow niche and HSC expansion deserve further investigation. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

SH2B3/LNK Loss of Function Promotes Atherosclerosis and Thrombosis

Human genome-wide association studies (GWAS) have revealed many novel genetic loci that are associated with coronary heart disease (CHD) but do not involve traditional risk factors. However, the relevant genes and mechanisms are largely unknown. One such locus resides in SH2B3/LNK, which is expressed in hematopoietic cells and suppresses thrombopoietin (TPO) signaling via its receptor (MPL). The common risk single nucleotide polymorphism (SNP) of SH2B3/LNK is associated with CHD, increased platelet and myeloid cell counts in peripheral blood and JAK2V617F positive myeloproliferative neoplasms.Analysis of human cord blood revealed that samples with TT risk SNP (R262W) in SH2B3/LNK was associated with expansion of hematopoietic stem cells (HSCs), increased MPL signaling in HSCs, increased megakaryopoiesis, and a paradoxical increased expression of LNK mRNA, indicating reduced LNK function but induction of LNK transcription downstream of increased MPL signaling. Since humans are hypercholesterolemic relative to mice, to assess the role of reduced LNK function in athero-thrombosis, we transplanted WT or Lnk-/- bone marrow (BM) cells into irradiated WT or low-density lipoprotein receptor knock-out (Ldlr-/-) recipient mice and fed the recipients with chow and western type diet (WTD), respectively. Although Lnk-/- BM recipients showed similarly increased platelet counts on both diets, we found that platelet activation and aggregation, formation of platelet/leukocyte aggregates, atherosclerosis and arterial thrombosis were markedly increased by the combination of hyperlipidemia and hematopoietic LNK deficiency. These diet-genotype interactions mirrored an increase in mean platelet volume, protease-activated receptor-4 (PAR4) agonist-induced P-selectin exposure on platelets and platelet protein kinase C (PKC) activity, suggesting increased platelet granule secretion. We also observed an increased number of bone marrow myeloid progenitor cells expressing elevated levels of the common-beta subunit (CBS) of the interleukin-3/granulocyte-macrophage colony-stimulating factor (IL-3/GM-CSF) receptor, which promotes leukocytosis and the formation of platelet/leukocyte aggregates in the bloodstream. The increased P-selectin exposure, platelet aggregation and PKC activity were largely reversed by high-density lipoprotein (HDL) or cyclodextrin, reagents that remove cholesterol from platelets. Consistently, cholesterol loading of platelets from the chow-fed mice led to increased P-selectin exposure and PKC activity upon PAR4 agonist stimulation but the increase was more pronounced in Lnk-/- platelets, indicating a cholesterol-genotype interaction. LNK deficiency also resulted in defective MPL-mediated TPO internalization in platelets, leading to increased plasma TPO levels in mice on both diets. Elevated TPO levels and enhanced MPL signaling likely acted together to increase the pool of HSCs and megakaryopoiesis, leading to marked thrombocytosis.These studies suggest that SH2B3/LNK loss of function variants lead to HSC expansion, overproduction of platelets, and in the setting of hypercholesterolemia, markedly increased myeloid cells, platelet activation and formation of platelet/leukocyte aggregates, leading to accelerated atherosclerosis and arterial thrombosis. Our studies point to the importance of treating dyslipidemia in the setting of platelet and myeloid cell overproduction, as observed in subjects with SH2B3/LNK risk alleles. DisclosuresLevine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy. Tall:Amgen: Consultancy; Arisaph: Consultancy; CSL: Consultancy.

Aquation Is a Crucial Activation Step for Anticancer Action of Ruthenium(II) Polypyridyl Complexes to Trigger Cancer Cell Apoptosis.

Aquation has been proposed as crucial chemical action step for ruthenium (Ru) complexes, but its effects on the action mechanisms remain elusive. Herein, we have demonstrated the aquation process of a potent Ru polypyridyl complex (RuBmp=[Ru(II) (bmbp)(phen)Cl]ClO4 , bmbp=2,6-bis(6-methylbenzimidazol-2-yl) pyridine, phen=phenanthroline) with a chloride ligand, and revealed that aquation of RuBmp effectively enhanced its hydrophilicity and cellular uptake, thus significantly increasing its anticancer efficacy. The aquation products (H-RuBmp=[Ru(II) (bmbp)(phen)Cl]ClO4 , [Ru(II) (bmbp)(phen)(H2 O)]ClO4 , bmbp) exhibited a much higher apoptosis-inducing ability than the intact complex, with involvement of caspase activation, mitochondria dysfunction, and interaction with cell membrane death receptors. H-RuBmp demonstrated a higher interaction potency with the cell membrane and induced higher levels of ROS overproduction in cancer cells to regulate the AKT, MAPK, and p53 signaling pathways. Taken together, this study could provide useful information for fine-tuning the rational design of next-generation metal medicines.

In vivo overactivation of the Notch signaling pathway in the developing cochlear epithelium

Notch signaling is thought to play important roles in both prosensory domain specification and cell fate determination during inner ear development. Inhibition of the Notch signaling pathway in prosensory cells results in excessive hair cell formation, while activation of the Notch signaling pathway by overexpression of activated Notch1 (Notch1-intercellular domain, NICD) in the cochlear epithelium results in ectopic sensory patches where NICD is expressed. However, the effect of Notch activation on the prosensory domain is not fully understood. To elucidate the precise roles of Notch signaling in cochlear prosensory epithelium we examined the effects of Notch overactivation on cochlear prosensory cells of transgenic mice with conditional NICD expression. The histology of the cochlear epithelium was investigated in these mice. The cochlear duct of conditional NICD embryos was wide and short, and the epithelium formed an abnormal tubular structure. Hair cell numbers were reduced though some hair cells developed where NICD was overexpressed. The decrease in hair cells was not accompanied by Hes5-positive and Prox1-positive supporting cell overproduction. Ectopic expression of early prosensory markers, such as Jag1 and Hes/Hey genes, was observed but no expression of Hes5 was found. Our data shows that NICD overexpression disrupts the extension of cochlear epithelium, and reduces the total numbers of hair cells and supporting cells in the sensory epithelium. Thus, an appropriate level of Notch signaling is needed for the normal extension of the cochlear epithelium and for differentiation of both hair cells and supporting cells.

Omega-3 PUFA docosahexaenoic acid decreases LPS-stimulated MUC5AC production by altering EGFR-related signaling in NCI-H292 cells

Chronic obstructive pulmonary disease (COPD) is an inflammatory process characterized by airway mucus hypersecretion. Lipopolysaccharides (LPS) are known to stimulate the production of mucin 5AC (MUC5AC) via epidermal growth factor receptor (EGFR) in human airway cells. Noteworthy, we have previously demonstrated that EGFR/Rac1/reactive oxygen species (ROS)/matrix metalloproteinase 9 (MMP-9) is a key signaling cascade regulating MUC5AC production in airway cells challenged with LPS.Various reports have shown an inverse association between the intake of polyunsaturated fatty acids (PUFA) of the n-3 (omega-3) family or fish consumption and COPD. In the present study, we investigated the influence of docosahexaenoic acid (DHA), one of the most important omega-3 PUFA contained in fish oil, on the production of MUC5AC in LPS-challenged human airway cells NCI–-H292. Our results indicate that DHA is capable of counteracting MUC5AC overproduction in LPS-stimulated cells by abrogating both EGFR phosphorylation and its downstream signaling pathway. This signaling pathway not only includes Rac1, ROS and MMP-9, but also NF-κB, since we have found that ROS require NF-κB activity to induce MMP-9 secretion and activation.

Indole-3-carbinol as inhibitors of glucocorticoid-induced apoptosis in osteoblastic cells through blocking ROS-mediated Nrf2 pathway

Apoptosis of osteoblasts induced by glucocorticoid (GC) has been identified as a main cause of osteoporosis, bone loss and fractures, and the oxidative stress was found as an important contributor. Therefore, natural or synthetic agents with antioxidant activities can antagonize GCs-induced apoptosis in osteoblasts, and thus demonstrate the potential application to reverse osteoporosis. In this study, we showed that, indole-3-carbinol (I3C), a natural product found in broadly consumed plants of the Brassica genus, could block the cytotoxic effects of dexamethasone (Dex), and elucidated the underlying molecular mechanisms. Firstly, we showed that, I3C could effectively suppress Dex-induced cytotoxicity and apoptotic cell death in osteoblastic cells, as evidenced by the decrease in Sub-G1 cell population. Treatment of the cells with Dex resulted in activation of caspase-3/-8/-9 and subsequent cleavage of PARP, which was also effectively blocked by co-incubation of I3C. Moreover, exposure to Dex triggered a rapid onset and time-dependent superoxide overproduction in osteoblastic cells, which was effectively suppressed by addition of I3C. Excess intracellular ROS induced by Dex significantly suppressed the expression levels of Nrf2 and the downstream effectors, HO1 and NQO1, but these changes could be reversed by I3C. Knockdown of Nrf2 using siRNA silencing technique significantly reversed the protective effects of I3C against Dex-induced apoptosis and ROS generation. Taken together, I3C can reverse cytotoxicity of Dex through blocking ROS overproduction and enhancement of Nrf2 expression. This study may provide a safe and good strategy for molecular intervention of GCs-induced osteoporosis by using natural products.

Parkin maintains mitochondrial levels of the protective Parkinson's disease-related enzyme 17-β hydroxysteroid dehydrogenase type 10.

Mutations of the PARK2 and PINK1 genes, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, respectively, cause autosomal recessive early-onset Parkinson's disease (PD). Parkin and PINK1 cooperate in a biochemical mitochondrial quality control pathway regulating mitochondrial morphology, dynamics and clearance. This study identifies the multifunctional PD-related mitochondrial matrix enzyme 17-β hydroxysteroid dehydrogenase type 10 (HSD17B10) as a new Parkin substrate. Parkin overproduction in cells increased mitochondrial HSD17B10 abundance by a mechanism involving ubiquitin chain extension, whereas PARK2 downregulation or deficiency caused mitochondrial HSD17B10 depletion in cells and mice. HSD17B10 levels were also found to be low in the brains of PD patients with PARK2 mutations. Confocal and Förster resonance energy transfer (FRET) microscopy revealed that HSD17B10 recruited Parkin to the translocase of the outer membrane (TOM), close to PINK1, both in functional mitochondria and after the collapse of mitochondrial membrane potential (ΔΨm). PD-causing PARK2 mutations impaired interaction with HSD17B10 and the HSD17B10-dependent mitochondrial translocation of Parkin. HSD17B10 overproduction promoted mitochondrial elongation and mitigated CCCP-induced mitochondrial degradation independently of enzymatic activity. These effects were abolished by overproduction of the fission-promiting dynamin-related protein 1 (Drp1). By contrast, siRNA-mediated HSD17B10 silencing enhanced mitochondrial fission and mitophagy. These findings suggest that the maintenance of appropriate mitochondrial HSD17B10 levels is one of the mechanisms by which Parkin preserves mitochondrial quality. The loss of this protective mechanism may contribute to mitochondrial dysfunction and neuronal degeneration in autosomal recessive PD.

Interleukin-10 inhibits autonomous myelopoiesis in patients with myelofibrosis.

The spontaneous formation of colony-forming units granulocyte/macrophage (CFU-GM) in semisolid cultures has been shown to be due to the endogenous release of cytokines and/or to the hypersensitivity of cells against growth factors. We have reported that increased autonomous CFU-GM growth is an in vitro characteristic of myelofibrosis (MF) which may reflect aberrant hematopoiesis in vivo. Because of its cytokine synthesis-inhibiting action, we speculated that interleukin-10 (IL-10) may inhibit pathological overproduction of myeloid cells in MF by suppression of autonomous myelopoiesis. In this study, IL-10 significantly inhibited autonomous CFU-GM formation in vitro from peripheral blood mononuclear cells (PB MNC) in 10 of 11 patients with MF tested. In all patients, there was a mean inhibition of 69% ranging from 35% to 100%. Suppression of autonomous CFU-GM formation by IL-10 was dose dependent and reversible by the addition of anti-IL-10 antibodies. Our results indicate that IL-10 is a potentially useful molecule to affect aberrant myelopoiesis in patients with MF.

What cell death does in development.

Cell death is prominent in gametogenesis and shapes and sculpts embryos. In non-mammalian embryos one sees little or no cell death prior to the maternal-zygotic transition, but, in mammalian embryos, characteristic deaths of one or two cells occur at the end of compaction and are apparently necessary for the separation of the trophoblast from the inner cell mass. Considerable sculpting of the embryo occurs by cell deaths during organogenesis, and appropriate cell numbers, especially in the CNS and in the immune system, are generated by massive overproduction of cells and selection of a few, with death of the rest. The timing, identity, and genetic control of specific cells that die have been well documented in Caenorhabditis, but in other embryos the stochastic nature of the deaths limit our ability to do more than identify the regions in which cells will die. Complete disruption of the cell death machinery can be lethal, but many mutations of the regulatory machinery yield only modest or no phenotypes, indicating substantial redundancy and compensation of regulatory mechanisms. Most of the deaths are apoptotic and are identified by techniques used to recognize apoptosis, but techniques identifying lysosomes (whether in dying or involuting cells or in the phagocytes that invade the tissue) also reveal patterns of cell death. Aberrant cell deaths that produce known phenotypes are typically localized, indicating that the mechanism of activating a programmed death in a specific region, rather than the mechanism of death, is aberrant. These results lead us to conclude that we need to know much more about the conversations among cells that lead cells to commit suicide.

Mutations of Calreticulin in Myeloproliferative Neoplasms Contribute to Disease Progression Not through Inhibition of Phagocytosis, but through Enhanced Proliferation Production of Myeloid Myelo-Erythroid Progenitor Cells

Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell disorders characterized by overproduction of mature myeloid cells. Recently, somatic mutation of calreticulin (CALR) was frequently found in MPN patients who do not have JAK2 mutation. The CALR mutation in MPN patients usually resulted in loss-of-function of CALR, which may induce impairment of physiological phagocytotic pathway, because surface CALR plays a critical role for macrophages in recognition of low-density lipoprotein receptor-related protein 1 (LRP1) on the targets, mediating pro-phagocytic signals.We hypothesized that the non-functional CALR mutation renders cells resistant to phagocytosis, and impairs the “programmed cell removal” of progenitors or mature blood cells, resulting in accumulation of hematopoietic cells in MPNs. In 135 Japanese MPNs patients enrolled in this study, including polycythemia vera (PV), essential thrombocytosis (ET) or primary myelofibrosis (PMF), 34 patients (25.2%) had CALR mutations, and 80 (59.3%) patients had JAK2 V617F mutation, respectively. CALR mutations were heterozygous in all 34 patients (27 patients with ET, 7 with PMF). On the other hand, JAK2 V617F mutations were found in 26 patients with PV, 39 with ET, and 15 with PMF.The expression levels of pro-phagocytotic CALR were normal in these MPN patients. We then performed in vitro phagocytosis assay to test whether the heterozygous CALR mutation affects engulfment of blood cells by macrophages. Hematopoietic stem cells (HSCs), progenitor cell populations such as common myeloid progenitors (CMPs), megakaryocyte/erythroid progenitors (MEPs) and granulocyte/monocyte progenitors (GMPs), and mature myeloid cells were isolated and opsonized, and were co-cultured with activated macrophages for 2 hours. After the culture, we enumerate macrophages and engulfed cells to analyze phagocytosis index (number of engulfed cells/number of macrophages) (Kuriyama et al. Blood 2012). However, the phagocytosis index was not changed in any of purified hematopoietic cells, irrespective of the presence of CALR or JAK2 mutation. These results strongly suggest that heterozygous, non-functional CALR mutation, and gain-of-function JAK2 mutations should not affect the engulfment process for hematopoietic cells by macrophages.We then investigated the effect of CALR or JAK2 mutations on differentiation and proliferation of stem or progenitor cells in MPNs. We performed colony-forming cell assay of multipotent cells, such as HSCs and CMPs, and evaluated clonal burden of CALR and JAK2 mutations in colonies derived from these stem and progenitor cells. In vitro culture showed that HSCs and CMPs with CALR and JAK2 mutations gave rise to granulocyte/monocyte (GM) or megakaryocyte/erythroid (MegE)-related colonies, whose frequencies were almost identical to those in wild-type controls, suggesting that these mutations do not affect myelo-erythroid lineage commitment at the multipotent stem or progenitor stages. In contrast, when we cultured GMPs and MEPs, frequencies of colonies with CALR or JAK2 mutations were significantly higher as compared to those in HSCs or CMPs (P<0.05); In patients with CALR mutation, 32.5% of HSC-derived colonies had CALR mutations, whereas in MEPs and GMPs, CALR mutations were found in 51.0% and 70%, respectively. In JAK2 mutated MPNs, 17.2% of HSC-derived colonies had JAK2 mutation, whereas 64.7% of MEP- and 87.9% of GMP-derived colonies had this mutation. These results indicate that clones with CALR or JAK2 mutations could contribute more robustly to maintain MEPs and GMPs, and these committed progenitors with mutations might produce higher amounts of mature myelo-erythroid cells, leading to progression of MPNs.Thus, CALR mutation contributes to progression of MPN, not through inhibition of phagocytic clearance, but presumably through enhanced production of myelo-erythroid lineage cells, as JAK2 mutation does. It is important to investigate the mechanism on which the CALR mutation causes overproduction of myelo-erythroid cells in future study. DisclosuresNo relevant conflicts of interest to declare.

Use of Minihepcidins As a “Medical Phlebotomy” in the Treatment of Polycythemia Vera

Polycythemia Vera (PV) is a myeloproliferative disorder in which the rate of erythropoiesis is massively increased. Most of the health concerns associated with PV are caused by increased red cell production, leading to increased risk of thrombosis due to red cell sludging in small vessels. PV is caused by JAK2 tyrosine kinase mutations that cause unregulated activation of the erythropoietin signaling pathway and massive overproduction of red cells. PV shows characteristic features of stress erythropoiesis, which is highly dependent on iron absorption and erythroid iron intake. The mainstay of therapy is therapeutic phlebotomy, the goal of which is to reduce the hematocrit (Hct) to 45% or less to minimize the risk of thrombosis. Since phlebotomy has no effect on bone marrow production of red cells the effect is transient and dependent on the development of an iron-deficient state for control of Hct. Poor compliance with phlebotomy and the intermittent nature of the intervention frequently leads to treatment failure. Iron deficiency or erythroid iron restriction may be effective in the treatment of PV by causing inhibition of erythropoietin signaling downstream of JAK2, mediated by inhibition of aconitase. We have previously shown that increased hepcidin level reduces transferrin saturation and erythroid iron intake and has a beneficial effect on the number of erythroid progenitors in a mouse model of Thalassemia Intermedia. Hepcidin mimetic peptides (minihepcidins (MH)) which replicate the biological effects of hepcidin may be beneficial in the treatment of PV by limiting iron availability to the developing erythrocyte, thereby overriding JAK2 stimulated erythropoiesis and reducing erythrocyte production.A mouse model of PV was generated by crossing a floxed heterozygous Jak2V617Ffl/fl (V617F is the most common mutation causing human PV) mouse with a mouse hemizygous for the Vav-iCre transgene expressed in hematopoietic cells. We generated a cohort of mice with the disease by engrafting Jak2V617Ffl/fl-Vav-iCre bone marrow into wild type (WT) mice. M009, which is a potent MH that decreases ferroportin expression in vitro and serum iron in vivo was administered subcutaneously at a supramaximal dose of 400 µg/mouse twice weekly. A high dose of M009 was used to ensure that a significant level of iron restriction was present throughout the study. In PK/PD studies in the rat equivalent doses of M009 had previously been shown to reduce serum iron by >80% for 48-72h post-dose. MH treatment for six weeks or vehicle control was initiated 2 months after bone marrow transplant, when the phenotype was fully established.Hct was 62.5 ± 4.3% (mean ± SD) in controls (n=5) at baseline, rising to 85.0 ± 4.0% at the end of study. In contrast in M009 treated animals Hct dropped from 69.3 ± 4.3% (n=6) to 38.8 ± 7.4%. Interim evaluation at 3 weeks indicated that the effect of M009 was evident early in the treatment period (Hct reduction of 36% in M009 treated animals versus 17% increase in vehicle controls). The effect of M009 on Hct was predominantly caused by a decrease in RBC (-34%) rather than MCV (-14%).These data indicate that MH-induced iron restriction can rapidly reduce the rate of erythropoiesis in this mouse model of human PV, leading to large reductions in Hct that would be highly clinically relevant if replicated in clinical trials. This effect was observed despite the fact that Hct continued to increase in control animals, indicative of even greater underlying disease activity during the treatment period. The dose of M009 that was used was high and probably supratherapeutic since circulating Hb was reduced below the level observed in WT mice. Nevertheless these results suggest that MH may be considered for development as a “medical phlebotomy” to provide continuous and improved control of accelerated erythropoiesis in the treatment of PV. DisclosuresCasu:Merganser Biotech LLC: Employment; Isis Pharmaceuticals, Inc.: Employment. Nemeth:Merganser Biotech LLC: Stockholders Other. Ganz:Merganser Biotech LLC: Stockholders Other. MacDonald:Merganser Biotech LLC: Employment, Equity Ownership. Rivella:merganser Biotech LLC: Consultancy, Research Funding, Stock options, Stock options Other; bayer: Consultancy, Research Funding; isis Pharmaceuticals, Inc.: Consultancy, Research Funding.

Targeting Glutamine Metabolism in Myeloproliferative Neoplasms

Introduction:The chronic Philadelphia chromosome negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are clonal stem cell disorders characterized by unregulated stem cell expansion and overproduction of blood cells. The acquired mutation JAK2V617F plays a central role in the pathogenesis of MPN. Ruxolitinib was the first FDA-approved JAK2 inhibitor for the treatment of MPNs; however, its use is limited by various toxicities due to its suppression of normal marrow function. Because its toxicity is related to dosage, combinations of drugs may enhance the therapeutic effectiveness of Ruxolitinib and permit the administration of lower doses of Ruxolitinib.Cancer cells depend on a continued supply of glucose and glutamine for cell survival and proliferation. Glutaminase (GLS) is the key enzyme in glutamine metabolism. We report here that JAK2V617F mutation is associated with increased cellular metabolism and the GLS inhibitor BPTES (Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide) improves the therapeutic efficacy of Ruxolitinib in both JAK2V617F-mutant cell lines and MPN patient samples.Methods:The JAK2V617F-mutant human erythroleukemia (HEL) cell line and the murine prolymphoid cell line Ba/F3 expressing wild-type or mutant JAK2 (BaF3-hEPOR-JAK2wt cell and BaF3-hEPOR-JAK2V617F cell) were maintained as previously described. Cells were treated with DMSO, BPTES (Sigma¨), and/or Ruxolitinib (Selleckchem¨) and cell proliferation was assessed by counting bright live cells in a hemocytometer using the trypan blue exclusion method. Mitochondrial respiration (oxygen consumption rate, OCR) and glycolysis (extracelluar acidification rate, ECAR) were measured using a Seahorse¨ XFe96 Bioanalyzer. We used 13C stable isotope analysis to determine differences in glucose and glutamine utilization. BaF3-hEPOR-JAK2wt cell and BaF3-hEPOR-JAK2V617F cell were grown in [U6]-13C glucose or [U5]-13C glutamine for 24 hours and metabolites extracted and prepared for GC-MS analysis. Data were analyzed using MassHunter software (Agilent Technologies¨).MPN patient specimens were obtained with informed consent and approval by the IRB of Northport VA Medical Center. Peripheral blood CD34+ cells from MPN patients were isolated using immunomagnetic beads (Miltenyi¨) as we previously did. MPN CD34+ cells were treated with DMSO, BPTES, and/or Ruxolitinib and assayed in methylcellulose semisolid medium (Stem Cell Technologies¨) with growth factors. Colonies were counted after 14 days of incubation.Results:OCR and ECAR were increased by 55% (p < 0.0001) and 2-fold (p < 0.0001), respectively, in BaF3-hEPOR-JAK2V617F cells compared to BaF3-hEPOR-JAK2wt cells. This indicated that the JAK2V617F-mutant cells have higher metabolic activity than JAK2WT cells. Stable isotope analysis using GC/MS showed that JAK2V617F-mutant cells had increased utilization of glucose and glutamine into the TCA cycle as well as increased lactate production from glucose. There was a higher glutaminase activity in the JAK2V617F-mutant cells than the JAK2WT cells.Next we determined the effect of the glutaminase inhibitor, BPTES, on Ruxolitinib-mediated inhibition of cell proliferation in JAK2V617F-mutant cell lines. While BPTES (5µM) had little effect on HEL cell growth, the combination of BPTES (5µM) and Ruxolitinib (1.5µM) significantly inhibited HEL cell growth by 66% while Ruxolitinib alone inhibited cell growth by only 35% (p = 0.04). Similar results were obtained with the BaF3-hEPOR-JAK2V617F cells. (Figure 1)Finally, we tested the combination therapy of BPTES and Ruxolitinib in peripheral blood CD34+ cells from 4 MPN patients (2 PMF and 2 ET). The combination of BPTES (2uM) and Ruxolitinib (125nM) suppressed total hematopoietic colony growth of MPN CD34+ cells by 91% in comparison to 68% inhibition by Ruxolitinib alone (p = 0.007). Both CFU-GM colony and BFUe colony were suppressed by the combination therapy more than by Ruxolitinib alone although it was only significant for CFU-GM colony formation. (Figure 2)Conclusion:Our study showed that JAK2V617F mutation was associated with increased cellular metabolism. Importantly, a glutaminase inhibitor enhanced the therapeutic efficacy of Ruxolitinib in both JAK2V617F-mutant cell lines and MPN patient peripheral blood CD34+ cells. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reduced TET2 function leads to T-cell lymphoma with follicular helper T-cell-like features in mice.

TET2 (Ten Eleven Translocation 2) is a dioxygenase that converts methylcytosine (mC) to hydroxymethylcytosine (hmC). TET2 loss-of-function mutations are highly frequent in subtypes of T-cell lymphoma that harbor follicular helper T (Tfh)-cell-like features, such as angioimmunoblastic T-cell lymphoma (30–83%) or peripheral T-cell lymphoma, not otherwise specified (10–49%), as well as myeloid malignancies. Here, we show that middle-aged Tet2 knockdown (Tet2gt/gt) mice exhibit Tfh-like cell overproduction in the spleen compared with control mice. The Tet2 knockdown mice eventually develop T-cell lymphoma with Tfh-like features after a long latency (median 67 weeks). Transcriptome analysis revealed that these lymphoma cells had Tfh-like gene expression patterns when compared with splenic CD4-positive cells of wild-type mice. The lymphoma cells showed lower hmC densities around the transcription start site (TSS) and higher mC densities at the regions of the TSS, gene body and CpG islands. These epigenetic changes, seen in Tet2 insufficiency-triggered lymphoma, possibly contributed to predated outgrowth of Tfh-like cells and subsequent lymphomagenesis. The mouse model described here suggests that TET2 mutations play a major role in the development of T-cell lymphoma with Tfh-like features in humans.