FLT3-Mediated MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis.

Abstract Abstract 963 Introduction: Flt3, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis. Primary myelofibrosis (PMF) is a Ph-negative (Ph−) myeloproliferative neoplasm (MPN) characterized by a myeloproliferation with increased hematopoietic progenitors (HPs) and a prominent proliferation of “dystrophic” megakaryocytes (MK). The JAK2V617F mutation is present in about 50% of PMF patients. Previous results from our group have revealed a MAPK pathway gene deregulation associated with an Flt3 transcript modulation in PMF patients. Since activation of Flt3 receptor is known to activate MAPK pathway, which plays a role in megakaryopoiesis, we studied the functional impact of MAPK and Flt3 abnormalities on PMF dysmegakaryopoiesis. Patients and Methods: The study included a group of 106 PMF patients. Transcriptome and QRT-PCR studies were performed on MACS selected CD34+ HPs, megakaryocytes (MK)-derived from CD34+ cell cultures and peripheral blood mononuclear cells (PBMNC) from PMF patients and healthy donors. Cell phenotype associated with Flt3 expression as well as phosphorylation levels of Flt3 and MAPK effectors were analyzed by flow cytometry. Functional studies (FL-induced stimulation and migration) were performed on MK-precursors at day 6 of CD34+ culture. Effect of i) MAPK inhibitors: PD98059, targeting ERK1/2; SB202190, SB203580, PD169316, targeting p38 and, SP600129, targeting JNK, ii) Flt3 inhibitors and iii) Flt3 monoclonal antibody was tested on PMF MK cell cultures. MAPK-induced transcripts were quantified by QRT-PCR in MK-precursors during an 18-hour FL-stimulation kinetic. FL mRNA level was evaluated by using QRT-PCR in bone marrow stromal cells and FL protein was quantified in plasma by using ELISA. Results: Comparative transcriptome analysis of CD34+ HPs and MK cells from PMF patients (with or without JAK2 mutation) and healthy donors showed that the MAPK pathway gene deregulation was independent of the presence of the JAK2V617F mutation. This alteration was associated with a modulation of mRNA Flt3 level in both types of cells. PMF patients also had a higher proportion of circulating Flt3+CD34+CD41+ cells as compared to healthy donors. This population demonstrated an increased phosphorylation of Flt3 on tyr591 and of MAPK (p38, p42/p44, JNK). MAPK effector phosphorylation was also increased in PMF CD34+ cells and MK-derived from CD34+ cell cultures, independently of JAK2 mutational status. In contrast to healthy donors, Flt3 membrane expression was maintained at all stages of in vitro megakaryocyte differentiation in PMF patients. The FL level was increased in the plasma of patients and was mainly expressed by bone marrow stromal cells. In contrast to healthy donors, in MK-derived from PMF CD34+ cell cultures, activation of Flt3/FL axis by addition of exogenous FL induced a MAPK hyper-phosphorylation, especially of p38 and p42/p44 as well as an up-regulation of downstream p38 transcripts (ATF-2, NFATC4, p53, AP-1, IL-8). Addition of chemical inhibitors targeting either MAPK or Flt3 and of an antibody directed against Flt3 reduced the phosphorylation of p38 and of its pathway effectors (MKK3/MKK6, MSK1, ATF2, HSP27 and MAPKAPK2) and normalized the PMF altered megakaryopoiesis. Lastly, in contrast to healthy donors, MK-derived from PMF CD34+ cells showed a FL-induced migration that was reversed by addition of p38αβ inhibitors. Conclusion: Our results demonstrated an increase in the FL circulating level in PMF patients that was mainly secreted by stromal cells. This was associated with an aberrant expression of Flt3 in CD34+ and MK cells and an alteration of the MAPK pathway activation in patients, independent of their JAK2 mutational status. The persistence of Flt3-mediated MAPK activation that participates in the PMF dysmegakaryopoiesis, suggests that drugs targeting “FL/Flt3-MAPK” axis could be promising agents for rescuing the altered megakaryopoiesis observed in patients. Our demonstration that FL, a cytokine mainly produced by stromal cells, participates in the altered megakaryopoiesis in PMF patients strengthens the hypothesis highlighting the crucial role of stroma cells in the hematopoietic deregulation that characterizes the disease. Disclosures: No relevant conflicts of interest to declare.

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FLT3 Mediated MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Blood ◽

10.1182/blood.v110.11.1532.1532 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1532-1532

Author(s):

Christophe Desterke ◽

Hans Hasselbalch ◽

Dominique Bordessoule ◽

Heinz Gisslinger ◽

Alessandro Vannucchi ◽

...

Keyword(s):

Gene Expression ◽

Cell Cultures ◽

Mononuclear Cells ◽

Mapk Pathway ◽

Primary Myelofibrosis ◽

Mapk Activation ◽

Specific Chemical ◽

Mutational Status ◽

Healthy Donors ◽

Cd34 Cells

Abstract Myeloproliferation, myelofibrosis, osteosclerosis and neo-angiogenesis are the major intrinsic pathophysiological features of Primary Myelofibrosis (PMF). The myeloproliferation is characterized by an increased number of circulating CD34+ cells with the prominent amplification of “dystrophic” megakaryocytes (MK) through to be responsible for myelofibrosis thought fibrogenic factor release. Comparison of CD34+ and MK cell gene expression profiling between PMF patients and healthy donors revealed a global deregulation of the MAPK pathway genes. This alteration is associated with a modulation of the FLT3 tyrosine kinase gene expression in CD34+ and MK cells from patients, independently of the JAK2V617F mutation presence. Quantification of the FLT3 transcript in mononuclear cells from patients with Polycythemia Vera and Essential Thrombocythemia showed that this over expression is mainly observed in JAK2WT PMF patients. This is associated with a higher proportion of FLT3+CD34+CD41+ cells in the blood of patients. Analysis of FLT3 membrane expression in MK-derived CD34+ cultures revealed that its expression was maintained all along MK differentiation in patients in contrast to healthy donors. Such a higher expression of FLT3 is associated with an increased concentration of its ligand in the platelet rich plasma from patients, independently of their JAK2 mutational status. The role of FLT3 in the regulation of hematopoiesis incited us to analyse whether its alteration could take part in the myeloproliferation and dysmegakaryopoiesis that characterizes PMF. A flow cytometry analysis of FLT3-downstream MAPK activation in PMF CD34+ cells showed a hyperphosphorylation of p38 and JNK as compared to CD34+ cells from normal blood. This phosphorylation was maintained in PMF MK-derived CD34+ cells at day 10. Addition of PD98059, a MAPK inhibitor, induced a dose dependent restoration of the in vitro megakaryopoiesis in PMF as shown by an increase in MK ploidy with apparition of 32N cells associated with a mature cytological aspect and an increase in CD41, CD42a and CD9 MK differentiation marker expression. PD98059 also increased the MK clonogenicity of CD34+ cells from all patients tested (5/5) as compared to healthy donors. Preliminary results using a specific chemical inhibitor of FLT3 in MK-derived CD34+ cell cultures reinforced the involvement of FLT3 in PMF MK differentiation. In presence of FLT3 ligand, the FLT3 mediated MAPK hyperphosphorylation in PMF MK cultures (D6) is reversed by either PD98059 or UO126, another ERK inhibitor and is accompanied by a slight increase in proliferative MK. This effect is not observed in MK cultures from normal CD34+ cells. Surprisingly, ligation of FLT3 by a monoclonal anti-FLT3 antibody in CD34+ cell cultures resulted in an increase MK proliferation. In conclusion, this work shows a deregulation of FLT3 and MAPK pathway in the PMF CD34+ cells and suggests that the persistence of the FLT3 mediated MAPK activation participates in the dysmegakaryopoiesis of PMF patients.

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The Tetraspanin CD9 Is Involved in Primary Myelofibrosis Dysmegakaryopoiesis Through c-Myb Regulation and Stroma Interactions,

Blood ◽

10.1182/blood.v118.21.3834.3834 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3834-3834

Author(s):

Christophe Desterke ◽

Costanza Bogani ◽

Lisa Pieri ◽

Alessandro M. Vannucchi ◽

Bernadette Guerton ◽

...

Keyword(s):

Bone Marrow ◽

Cell Migration ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Actin Polymerization ◽

Primary Myelofibrosis ◽

Membrane Expression ◽

Healthy Donors ◽

Cd34 Cells

Abstract Abstract 3834 Introduction: CD9, a four transmembrane glycoprotein belonging to the tetraspanin family, is suggested to regulate cell motility and adhesion and to play a role in megakaryopoiesis. It has been reported to be a molecular marker of primary myelofibrosis (PMF) being characterized by myeloproliferation, dysmegakaryopoiesis, alterated bone marrow/spleen stroma and extramedullary haematopoiesis. CD9 mRNA has been shown to be overexpressed in CD34+ PMF HPs and its membrane expression level was correlated with platelet counts. Our recent data evidencing an alteration of CD9 expression in PMF megakaryocytes (MK) have encouraged us to investigate whether CD9 participates in the dysmegakaryopoiesis and whether it is involved in the dialogue between MK and stromal cells in PMF patients. Patients and Methods: CD34+ cells were MACS selected from the peripheral blood of PMF patients (n=67) and of unmobilized healthy donors (n=61). Functional studies were performed on MK precursor-derived from CD34+ cells cultured in MK medium with ou without monoclonal antibodies (Syb mAb) or siRNAs targeting CD9. CXCL12-induced MK migration was performed in Boyden chambers. Bone marrow mesenchymal stromal cells (MSC) from healthy donors and PMF patients were cultured in DMEM+10%FCS. Results: Our results showed that CD9 membrane expression was altered on CD34+ cells and on MK precursor-derived from PMF CD34+ cells. Binding of CD9 with Syb mAb restored the in vitro megakaryocyte differentiation process that was altered in patients as shown by an increase in: i) megakaryocytic colony formation in semisolid medium, ii) CD41 and CD62p MK differentiation marker and GATA-1 expression, iii) MK cytoplasmic maturation, iv) apoptotic MK number (reduced AKT phosphorylation and Bcl-XL expression and increased percentage of Annexin+ cells). Activation of CD9 was also associated with regulation of MAPK and AKT-GSK3β pathways whose balance is involved in MK differentiation. Treatment of PMF MK precursors by Syb modulated activation of the MAPK pathway as shown by an increased of p38, JNK and GSK3β phosphorylation and of AP-1 mRNA expression. Taking into account the structure of the tetraspanin molecular network, binding with Syb mAb might also impact the effects associated to the multimolecular complex in which CD9 is involved. This prompted us to study the effects of a molecular silencing of CD9 on the PMF MK differentiation. We showed that, in contrast to the Syb mAb, addition of CD9 siRNA to PMF megakaryocytes reduced their transcriptional program including c-Myb, a transcription factor that is involved in CD9 regulation during megakaryopoiesis. Given the role of CD9 in cell migration, we further investigated whether it could be involved in the megakaryocytic precursor migration observed in patients. We showed that silencing CD9 reduced the CXCL12-dependent megakaryocytic precursor migration as well as the CXCR4 and CXCL12 transcription and that this migration involved actin polymerization. c-Myb siRNA restored CXCR4 and CXCL12 expression and reduced actin polymerization suggesting that CD9 was involved, via c-Myb, in the CXCL12-dependent megakaryocytic precursor migration. Effect of CD9 on cell migration is often interpreted as related to modulation of integrins participating in the integrin/tetraspanin network and of their interaction with mesenchymal stromal cells (MSC). We showed that several genes involving the CD9 partner interactome were over-expressed in MSC from PMF bone marrow as compared to MSC from healthy donors. Preliminary results showing that PMF MK precursors display different behaviour in terms of cell survival and adhesion when co-cultured on bone marrow MSC from PMF patients as compared to healthy donors suggest that interactions between MKs and bone marrow MSC is involved in PMF dysmegakaryopoiesis. Addition of Syb reverses these alterations suggesting the participation of CD9 in the abnormal dialogue between MK and MSC. Conclusion: Our results show a deregulation of CD9 expression in megakaryocytes from PMF patients. They also suggest that CD9 i) participates in PMF dysmegakaryopoieis in terms of MK differentiation and survival and ii) is involved in the increased MK precursor migration through alterations of the CXCL12/CXCR4 axis. Our data further support the role of bone marrow stroma in PMF dysmegakaryopoeisis through CD9 interactions. Disclosures: No relevant conflicts of interest to declare.

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Combined Disruption of Both the MEK/ERK and the IL-6R/STAT3 Pathways Is Required To Induce Apoptosis of Multiple Myeloma Cells in the Presence of Bone Marrow Stromal Cells.

Blood ◽

10.1182/blood.v104.11.4839.4839 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4839-4839

Author(s):

Manik Chatterjee ◽

Thorsten Stuehmer ◽

Pia Herrmann ◽

Kurt Bommert ◽

Bernd Dorken ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Mapk Pathway ◽

Underlying Mechanism ◽

Marrow Stromal Cells ◽

Erk Pathway ◽

Stat3 Pathway ◽

Survival Effect

Abstract The IL-6R/STAT3 pathway has been reported to critically contribute to the pathogenesis of multiple myeloma (MM) and to protect MM cells from apoptosis. However, recently we could demonstrate that MM cells become independent of the IL-6R/STAT3 pathway if they are cocultured with bone marrow stromal cells (BMSCs), suggesting that the BM microenvironment stimulates IL-6-independent pathways that exert a pro-survival effect. It was therfore the aim of this study to analyze the underlying mechanism of this phenomenon. Pathway analysis revealed that BMSCs stimulate STAT3 via the IL-6R, and MAPK in parts via IL-6R-independent mechanisms. Abolition of MEK1, 2 activity with PD98059, or of ERK1,2 through siRNA constructs, was insufficient to induce apoptosis. However, the combined disruption of the IL-6R/STAT3 and MEK1,2/ERK1,2 pathways led to strong induction of apoptosis even in the presence of BMSCs. Thus, disruption of the MEK/ERK pathway restores IL-6/STAT3 dependence of MM cells in the presence of BMSCs indicating that BMSC-mediated induction of the MEK/MAPK pathway is the mechanism by which BMSCs render MM cells IL-6/STAT3 idependent. Consequently, in the presence of cells from the BM microenvironment the combined targeting of different (and independently activated) pathways is required to efficiently induce apoptosis of MM cells. This effect was observed with MM cell lines and with primary MM cells and might have direct implications for the development of future therapeutic strategies for MM.

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Muc1 Expression Identifies Human Self-Renewing Stem/Progenitor Populations and Is Elevated in AML CD34+ Cells.

Blood ◽

10.1182/blood.v110.11.4040.4040 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4040-4040

Author(s):

Szabolcs Fatrai ◽

Simon M.G.J. Daenen ◽

Edo Vellenga ◽

Jan J. Schuringa

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Fold Increase ◽

Marrow Stromal Cells ◽

Muc1 Expression ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Mucin1 (Muc1) is a membrane glycoprotein which is expressed on most of the normal secretory epithelial cells as well as on hematopoietic cells. It is involved in migration, adhesion and intracellular signalling. Muc1 can be cleaved close to the membrane-proximal region, resulting in an intracellular Muc1 that can associate with or activate various signalling pathway components such as b-catenin, p53 and HIF1a. Based on these properties, Muc1 expression was analysed in human hematopoietic stem/progenitor cells. Muc1 mRNA expression was highest in the immature CD34+/CD38− cells and was reduced upon maturation towards the progenitor stage. Cord blood (CB) CD34+ cells were sorted into Muc1+ and Muc1− populations followed by CFC and LTC-IC assays and these experiments revealed that the stem and progenitor cells reside predominantly in the CD34+/Muc1+ fraction. Importantly, we observed strongly increased Muc1 expression in the CD34+ subfraction of AML mononuclear cells. These results tempted us to further study the role of Muc1 overexpression in human CD34+ stem/progenitor cells. Full-length Muc1 (Muc1F) and a Muc1 isoform with a deleted extracellular domain (DTR) were stably expressed in CB CD34+ cells using a retroviral approach. Upon coculture with MS5 bone marrow stromal cells, a two-fold increase in expansion of suspension cells was observed in both Muc1F and DTR cultures. In line with these results, we observed an increase in progenitor counts in the Muc1F and DTR group as determined by CFC assays in methylcellulose. Upon replating of CFC cultures, Muc1F and DTR were giving rise to secondary colonies in contrast to empty vector control groups, indicating that self-renewal was imposed on progenitors by expression of Muc1. A 3-fold and 2-fold increase in stem cell frequencies was observed in the DTR and Muc1F groups, respectively, as determined by LTC-IC assays. To determine whether the above mentioned phenotypes in MS5 co-cultures were stroma-dependent, we expanded Muc1F and DTR-transduced cells in cytokine-driven liquid cultures. However, no proliferative advantage or increase in CFC frequencies was observed suggesting that Muc1 requires bone marrow stromal cells. In conclusion, our data indicate that HSCs as well as AML cells are enriched for Muc1 expression, and that overexpression of Muc1 in CB cells is sufficient to increase both progenitor and stem cell frequencies.

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MicroRNA Changes Occur in Multiple Myeloma Cells in the Context of Bone Marrow Milieu.

Blood ◽

10.1182/blood.v114.22.1785.1785 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1785-1785 ◽

Cited By ~ 1

Author(s):

Roccaro M. Roccaro ◽

Antonio Sacco ◽

Abdel Kareem Azab ◽

Feda Azab ◽

Hai Ngo ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Expression Patterns ◽

Mirna Gene ◽

Marrow Stromal Cells ◽

Stem Loop ◽

Mirna Profiling ◽

Qrt Pcr

Abstract Abstract 1785 Poster Board I-811 Background We and Others have previously demonstrated that primary multiple myeloma (MM) cells are characterized by a specific microRNA (miRNA) signature compared to the related normal plasmacell counterpart; and that miRNAs play a crucial role in regulating MM pathogenesis. Nevertheless, miRNA changes that occur in MM cells in the context of the bone marrow microenvironment have not been previously examined. Therefore, characterization of miRNA profiling of MM cells in conjunction with bone marrow stromal cells (BMSCs) is important to better understand the underlying molecular changes that lead to initiation and progression of this disease. Methods We performed miRNA-expression-profiling of MM cell lines (MM.1S; RPMI8226) that were co-cultured with primary BMSCs obtained from 5 MM patients, using liquid phase Luminex microbead miRNA profiling (Luminex, Austin, TX). The expression patterns of unfiltered data were performed using unsupervised hierarchical clustering of samples, based on centroid linkage and 1-correlation distance metric, using dChip (www.dchip.org). To further define those miRNAs differentially expressed between groups (patients vs normal), the data were filtered on significance of differences using ANOVA test, (P < 0.05). Microbead-miRNA profiling data were validated data by stem-loop qRT-PCR. To identify specific predicted miRNA-targeted mRNAs, TargetScan, PicTar, and miRanda algorithms were used. Results miRNA profiling of MM cells cultured with primary BMSCs (MM+BMSC system) differs from MM cells which were not grown in contact with primary BMSCs (MM cells alone). Specifically, we observed increased expression of miRNA-450, -432*, -299-5p, -409-3p, -29b, -542-5p, -184, -517*, -218, 128b, -142-5p and -211 (P<0.05) in MM cells obtained from the MM+BMSC system, compared to MM cells alone. Stem-loop qRT-PCR was performed on matched samples and showed expression patterns similar to those observed in miRNA analysis. Using algorithms commonly used to predict human miRNA gene targets (miRanda; TargetScan; PicTar), predicted targets of the increased miRNAs included negative regulators of NFkB, PI3K/Akt/mTOR, and MAPK/ERK signaling pathways, such as PTEN, KSR2, TWEAK, and DUSP; as well as tumor suppressors (MCC, TSSC1, TUSC1, FBW7, RHOBTB), pro-apoptotic factors and cyclin-dependent kinases inhibitors. These data demonstrate that bone marrow stromal cells exert a modulatory effect on miRNA profiling in MM cells, which results in promoting MM cell growth and reducing MM cell survival. Disclosures Ghobrial: Millennium : Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

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Comparative Global Transcriptome Analysis of Bone Marrow Stromal Cells (BMSC), Human Embryonic Stem (hES) Cells and CD34+ Cells.

Blood ◽

10.1182/blood.v114.22.36.36 ◽

2009 ◽

Vol 114 (22) ◽

pp. 36-36

Author(s):

Ping Jin ◽

Jiaqiang Ren ◽

Marianna Sabatino ◽

Arun Balakumaran ◽

Sergei Kuznetsov ◽

...

Keyword(s):

Stem Cell ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Healthy Subjects ◽

Immune Modulation ◽

Embryonic Stem ◽

Marrow Stromal Cells ◽

Cd34 Cells ◽

Global Transcriptome ◽

Tgf Β1

Abstract Abstract 36 Bone marrow stromal cells (BMSCs, also known as “mesenchymal stem cells) are being used to treat acute graft-versus-host-disease, but their mechanisms of immune modulation are not certain. In vitro studies suggest that the immunosuppressive activity of BMSCs involves multiple factors including transforming growth factor β (TGF-β), hepatocyte growth factor (HGF), TNF-α, IFN-ψ, IL-10, IL-2 and prostaglandin E2 (PGE2). In this study we compared BMSCs with other types of stem cells using global transcriptome and microRNA (miR) expression analysis to identify factors that might contribute to their immunosuppressive effects and to identify biomarkers for assessing the stability, consistency, comparability, and potency of clinical BMSC products. BMSCs (passage 2 or 3) made from marrow aspirates of 4 healthy subjects by culturing in flasks and cell factories with 20% fetal bovine serum (FBS) were compared to 3 human embryonic stem cell lines (hES) and CD34+ cells isolated from G-CSF-mobilized peripheral blood from 3 healthy subjects. The cells were analyzed with an miR expression array with more than 800 probes and an oligonucleotide expression microarray with more than 35,000 probes. Hierarchical clustering analysis of the miR expression data separated the 3 types of cells into 3 distinct groups with unique signatures. MiRNA implicated in cancer and stem cell development that were up-regulated in BMSC compared to CD34+ cells and hES included miR21 and 125b and cancer and stem cell miR down-regulated in BMSCs included miR106a, 106b, 18a, 19b and 20b. When compared to CD34+ cells, several miR in the onco-miR17-92 cluster (miR17, 18a, 20a, 19b-1, and 92-1) and onco-miR106a-363 cluster (106a, 18b, 20b, 19b-2, 92-2, and 363) were down-regulated in BMSCs. Hierarchical analysis of the 4,600 genes that were expressed in greater than 80% of samples and were increased more than 2-fold in at least one sample clustered the 3 cell types into separate groups. Ingenuity pathway analysis revealed that the following pathways contained a significant number of genes that were up-regulated in BMSCs compared to both CD34+ cells and hES: actin based motility by rho, actin cytoskeletal signaling, integrin signaling, androgen signaling, IL-8 signaling VEGF signaling, PTEN signaling, oncostatin M signaling, fMLP signaling in neutrophils, inositol metabolism, cavelolar-mediated endocytosis and NRF2-mediated oxidative stress response pathway. DNA methylation and transcription repression pathway genes were down-regulated in BMSCs. Analysis of specific differentially expressed genes found that the gene most up-regulated in BMSCs was TGF-β1. Its expression was 346-fold fold greater in BMSCs than CD34+ cells and 298-fold greater than in hES. In addition, when compared to CD34+ cells, the expression of IL-6 was up-regulated 13.6-fold in BMSCs, prostaglandin E synthase was up-regulated 13.2-fold, and HGF 8.43-fold. These results support a possible role for TGF-β1, IL-6, HGF and PGE2 in BMSC-mediated immune modulation. TGF-β1, IL-6, HGF and PGE2 synthase are potential BMSC potency biomarkers, but further studies, including the correlation of the expression of these biomarkers in specific BMSC products with the clinical outcomes of patients treated with these products, are needed. Disclosures: No relevant conflicts of interest to declare.

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Endogenous TGF-beta1 Mediated Osteogenic Impairment of Medullar Mesenchymal Stromal Cells in Primary Myelofibrosis

Blood ◽

10.1182/blood.v124.21.1873.1873 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1873-1873

Author(s):

Christophe Martinaud ◽

Christophe Desterke ◽

Johanna Konopacki ◽

Lisa Pieri ◽

Rachel Golub ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Hematopoietic Cells ◽

Primary Myelofibrosis ◽

Osteogenic Potential ◽

Healthy Donors

Abstract Primary myelofibrosis (PMF) is myeloproliferative neoplasm characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in bone marrow and spleen. Mesenchymal stromal cells (MSCs) are reported to play a pivotal role in fibrosis and stromal changes are considered as a reactive counterpart of the cytokine production by clonal hematopoietic cells. The present study shows that MSCs from patients demonstrate functional abnormalities that are unexpectedly maintained ex-vivo, in culture. Material and Methods: we studied MSCs and bone marrow sections from PMF patients (n=12) as compared to healthy donors (HDs) (n=6). We tested their proliferation, immunophenotype, hematopoiesis supporting capacities, differentiation abilities, in-vivo osteogenic assays, and performed secretome and transcriptome analysis. Results: We found that PMF-MSCs exhibit similar proliferative capacity and long-term hematopoiesis supporting abilities as compare to healthy donors. They overproduce interleukin 6, VEGF, RANTES, PDGF, BMP-2 and surprisingly TGF-beta1. MSCs from fibrotic PMF patients express high levels of glycosaminoglycans. Adipocytes and chondrocytes differentiation abilities were not different as compared to HDs but PMF-MSCs exhibit an increased in vitro potential. Implementation on scaffold in nude mice confirmed, in vivo, this increased osteogenic potential. We then looked into gene expression and discovered that PMF-MSCs show an original transcriptome signature related to osteogenic lineage and TGF-beta1. Indeed, osteogenic genes such as Runx2, Dlx5, Twist1, Noggin, Sclerostin, GDF5 and Serpine1 are deregulated and suggest a potential osteoprogenitor priming of PMF-MSCs. These molecular results also advocated for a TGF-beta1 impregnation that prompted us to study its impact on PMF-MSCs osteogenic differentiation. First, we then showed that Smad2 is intrinsically over-activated in PMF-MSC and that stimulation by TGF-beta1 is associated with an increase phospho-Smad2 level and an enhancement of bone master gene regulator Runx2 expression. Then, we inhibited TGF-beta1 pathway by by SB-431542 and evidenced a specific behavior of osteogenic MSCs differentiation in patients, suggesting involvement of TGF-beta1 in osteogenic impairment. Conclusion: Altogether, our results identify a signature of PMF-MSCs and suggest that they participate in PMF osteogenic dysregulation independently from in vivo local stimulation by clonal hematopoietic cells Disclosures No relevant conflicts of interest to declare.

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Interleukin-32 Induce Production of IL-6 in Multiple Myeloma Bone Marrow Stromal Cells

Blood ◽

10.1182/blood.v128.22.3253.3253 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3253-3253

Author(s):

Xuanru Lin ◽

Xing Guo ◽

Jing Chen ◽

Qingxiao Chen ◽

Enfan Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Cell Lines ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Inflammatory Factor ◽

Healthy Controls ◽

Qrt Pcr ◽

Stat Pathway

Abstract Background: Multiple myeloma (MM) is closely associated with inflammation. Patients with auto-immune disease、history of infection and other inflammatory disease have higher incidence of MM. IL-6 is the most important inflammatory factor in MM which plays a key role in the proliferation and progression. We previously demonstrated that MM cells were modified by bone marrow stromal cells (BMSCs) that formulate a inflammatory microenvironment in bone marrow (BM) and secret IL-6. How the inflammation makes BMSCs secret IL-6, however, remained undocumented. Our subsequent study compared the differential secretion of peripheral blood (PB) between MM patients and normal people by cytokine array, and showed that interleukin 32(IL-32) is highly expressed in MM patients. IL-32, also named natural killer 4(NK-4), is a newly found inflammatory factor. It was reported in solid tumors IL-32 is a pro-inflammatory factor which triggers a massive amplification of inflammatory process resulting in the change of other inflammatory factor including IL-6,IL-10,TNF-α. In this study, we examined BMSCs cytokines in MM BM and found that IL-32 was a functional factor in the process of inflammation in MM BM microenvironment. Results: First, to test our previous study, we detected IL-32 in BM supernatant and PB supernatant in both MM patients (n=45) and healthy controls (n=13) by ELISA. Result showed that in both BM and PB, MM patients have higher expression of IL-32 compared to healthy controls (P<0.05). Next, total BM cells(both CD138+ and CD138- cells) from MM patients were assayed by qRT-PCR for gene expression analysis.IL-32 were highly expressed in MM BM cells and the CD138+ cells (P<0.05). We also detected IL-32 in MM cell lines (RPMI 8226,OPM-2) and BMSCs isolated from MM patients by qRT-PCR, Western blot, and ELISA, and found that IL-32 was highly expressed in MM cell lines than BMSCs. In contrast, proteinase 3(PR3, receptor of IL-32) was highly expressed in BMSCs compared to MM cell lines. Second, we stimulated the MM BMSCs with recombinant IL-32α, and found that the secretion of IL-6,CCL3 (MIP1-α), CCL4(MIP-1β) were significantly increased and CCL-5(RANTES)and IL-10 were decreased (P<0.05). Further, Western blot was applied to detect the inflammation molecular pathway in BMSCs. JAK-STAT pathway and NF-κB pathway were activated, and the phosphorylation of STAT3 was increased. After we knock down the PR3 in BMSCs, these changes were reduced. We repeated these experiments in BMSCs isolated from 15 different MM patients, the phenomenon mentioned above showed in 11 patients. The recombinant IL-32α was also used to stimulate 8226 and OPM-2 cells, but these two kinds of MM cells didn't secret IL-6, and no significant change in cell proliferation or cell apoptosis. Finally, our group co-cultured the MM BMSCs with 8226 and OPM-2 cells. The secretion of IL-6 and the phosphorylation of JAK-STAT pathway in BMSCs were also increased. Knockdown of IL-32 in 8226 and OPM-2 cells weakened these changes.MM Cell proliferation and cell cycles after co-culture with MM BMSCs are under investigation. Conclusion: our findings suggest that IL-32 is mainly secreted by MM cells. It may not directly promote the MM cells to grow. However, IL-32 promote the MM BMSCs to secret more cytokines including IL-6,CCL3,CCL4 by activating the JAK-STAT3 pathway, which lead to a amplification of inflammation in BM environment, resulting in the cell proliferation . Disclosures No relevant conflicts of interest to declare.

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Alpha-Catenin, ARHGAP21 and β-Catenin Are Abnormally Expressed in Bone Marrow Cells From Patients with Myelodysplatic Syndromes and Are a Possible Target for Decitabine Treatment.

Blood ◽

10.1182/blood.v114.22.1778.1778 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1778-1778

Author(s):

Karin Spat Barcellos ◽

Sheila Maria Winnischofer ◽

Mariana Lazarini ◽

Adriana Silva Santos Duarte ◽

Carolina Louzao Bigarella ◽

...

Keyword(s):

Bone Marrow ◽

Cell Membrane ◽

Myelodysplastic Syndrome ◽

Stromal Cells ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Refractory Anemia ◽

Beta Catenin ◽

Healthy Donors ◽

Cd34 Cells

Abstract Abstract 1778 Poster Board I-804 Introduction Myelodysplastic syndrome (MDS) encloses a group of clonal hematopoietic disorders clinically and morphologically characterized by ineffective hematopoiesis. The gene encoding alpha-catenin (CTNNA1) is expressed at a much lower level in leukemia-initiating stem cells from individuals with MDS del(5q). Thus, loss of alpha-catenin tumor suppressor expression in hematopoietic cells may provide a growth advantage that collaborates MDS pathogenesis. ARHGAP21, a negative regulator of RhoGTPase signaling pathways, is a partner of alpha-catenin that controls its recruitment to the adherens junctions. ARHGAP21 is upregulated during myeloid differentiation, and could be involved in the malignant process of hematopoietic cells. In addition, alpha-catenin is a target for decitabine (DAC) treatment, a demethylating agent with potent antitumorigenic properties against MDS. The aim of this work was to evaluate the expression of alpha-catenin, ARHGAP21 and beta-catenin (gene CTNNB1) in bone marrow cells from MDS patients with or without del(5q) and to analyze CTNNA1, ARHGAP21 and CTNNB1 expression after DAC treatment. PATIENTS AND METHODS cells were isolated from bone marrow of 6 MDS patients, including 5 refractory anemia (RA), being two with del(5q), and 1 refractory anemia with excess blasts (RAEB), based on the French-American-British classification, and 4 control subjects (normal hematopoietic tissues were obtained from healthy donors). The study was approved by the National Ethical Committee Board and bone marrow samples were collected at the Hematology and Hemotherapy Center, University of Campinas, after all participants provided informed written consent. Alpha-catenin, ARHGAP21 and beta-catenin localization in CD34+ cells was obtained using confocal microscopic analysis. ARHGAP21 localization was also analyzed in HS-5 stromal cells that were submitted to a CTNNA1 RNA interference (RNAi) approach. Leukemia cells lines (HL-60 and P-39) and bone marrow mononuclear cells obtained from 7 MDS patients, 5 RA and 2 refractory anemia with ringed sideroblasts (RARS), were treated with DAC for 72 hours; then mRNA expression of CTNNA1, ARHGAP21 and CTNNB1 was analyzed by Real-time PCR (normalized by GAPDH and beta-actin). RESULTS alpha-catenin, ARHGAP21 and beta-catenin are preferentially localized in the nucleus of CD34+ cells from MDS patients in contrast to the preferential cytoplasm and membrane localization in healthy donors and in MDS patients with del(5q). In del(5q) patients and healthy donors, ARHGAP21 and alpha-catenin co-localizated in the cell membrane. ARHGAP21 was abnormally expressed in cells with decreased CTNNA1 expression: in HS-5 stromal cells, ARHGAP21 was localized at the cytoplasm (mainly in the perinuclear region) and at the nucleus, in contrast, ARHGAP21 was poorly detectable in the nucleus of CTNNA1-RNAi treated cells. DAC treatment of MDS cells and leukemia cell lines induced CTNNA1, ARHGAP21, and CTNNB1 expression in a dose-dependent way. In HL60 and P39 cells, ARHGAP21 relocate to the cell membrane after DAC treatment. CONCLUSION The abnormal localization of alpha-catenin, ARHGAP21 and beta-catenin in MDS may compromise the reorganization of actin dynamics at sites of cell–cell contact that stabilizes cadherin-mediated cell–cell adhesion; moreover, these results also suggest a deficient recruitment of alpha-catenin to the cell membrane and an aberrant signaling in the Wnt pathway. In addition, ARHGAP21, alpha-catenin and beta-catenin are a target for DAC treatment in MDS. Supported by: FAPESP. Keywords: alpha-catenin, ARHGAP21, beta-catenin, myelodysplastic syndrome, Rho-GAP, decitabine Disclosures No relevant conflicts of interest to declare.

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Aberrant Megakaryocyte Gene Expression Contributes to Primary Myelofibrosis.

Blood ◽

10.1182/blood.v120.21.2867.2867 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2867-2867

Author(s):

Laure Gilles ◽

Christy Finke ◽

Terra L Lasho ◽

Animesh Pardanani ◽

Ayalew Tefferi ◽

...

Keyword(s):

Gene Expression ◽

Clinical Trial ◽

Bone Marrow ◽

Flow Cytometry ◽

Primary Myelofibrosis ◽

Gene Set Enrichment Analysis ◽

Gene Expression Program ◽

Healthy Donors ◽

Cd34 Cells ◽

Expression Program

Abstract Abstract 2867 Primary myelofibrosis (PMF) is a clonal hematologic malignancy, which results from the transformation of a pluripotent hematopoietic progenitor cell. A major consequence of this transformation is increased hematopoiesis and an overproduction of abnormal blood cells. PMF is associated with bone marrow fibrosis, extramedullary hematopoiesis, increased numbers of circulating CD34+ cells, splenomegaly, and a propensity to evolve to AML. Patients also display anemia and thrombocytopenia and harbor abnormal, immature megakaryocytes (Mks) in their bone marrow and spleen. PMF patients can present well known mutations including JAK2V617F (65%), MPL (10%), TET2 (17%), CBL (6%), IDH (4%,), which are not specific to the disease and are also present in polycythemia vera, essential thrombocythemia and AML. We hypothesize that the genetic events associated with PMF, including MPL and JAK2 mutations, contribute to defects in Mk maturation, but that additional changes are needed to explain the striking abnormalities seen in PMF relative to the other myeloproliferative diseases. Although there have been studies to examine the aberrant gene expression program of CD34+ cells of PMF patients, we chose to examine the changes that occur in gene expression specifically in Mks as a way to better understand their abnormal differentiation and to determine their contribution to the disease. Primary CD34+ cells from PMF patients and healthy donors were cultivated in serum free media supplemented with recombinant TPO, BSA, liposomes, insulin and transferrin to support the growth of Mks. After 10 days of differentiation, we evaluated the cultures for proliferation, apoptosis and differentiation by flow cytometry. We found that PMF specimens gave rise to a lower percentage of mature (CD41+CD42+) cells as compared to healthy donors, but showed, a lower ploidy level, a greater proliferation and increased survival. These observations are consistent with the clinical observations that PMF bone marrow is characterized by an increased number of immature, dysplastic Mks. We used flow cytometry to collect two populations of cells for analysis: immature CD41+CD42− Mks, and CD41+CD42+ mature MKs. After sorting, we extracted RNA and performed whole genome microarray analysis with Illumina Human HT12-v4 arrays on cohorts of PMF and control specimens. Gene expression data were analyzed by GeneSpring and Gene Set Enrichment Analysis (GSEA). We found that the CD41+CD42− MKs derived from PMF progenitors showed reduced expression of GATA1 as compared to control cells, as expected based on previous study by Dr. Alessandro Vannuchi. GeneSpring analysis revealed that myeloid transcription factors, including CEBPa, GFI1, and SPI1 (PU.1), which are not expressed in normal MKs, are strikingly and significantly overexpressed in PMF samples. Moreover, c-myb, which regulates the erythroid/Mk cell fate decision, FOG-1 and AML1, are also overexpressed in PMF Mks. This aberrant myeloid gene expression program in PMF Mks is reminiscent of a similar defect we observed in Mks with reduced expression of GATA-1 and GATA-2. We predict that reduced levels of GATA-1 protein in PMF Mks, as reported by Dr. Alessandro Vannucchi and colleagues, is in part responsible for the aberrant growth and differentiation of the PMF Mks. Our data support the model that PMF Mks are defective in their ability to properly regulate expression of hematopoietic regulators. Further analysis by GSEA revealed that hematopoietic and cytokine pathways are among those that are highly enriched in PMF Mks. We recently reported that the molecules dimethylfasudil (diMF) and MLN9237 are able to selectively increase ploidy, Mk surface marker expression, and apoptosis of malignant Mks. We treated Mks derived from PMF progenitor cells with diMF and observed a high increase in polyploidization accompanied with a reduction of Mks proliferation. Thus, diMF is able to partially restore Mk differentiation of PMF cells, supporting the testing of polyploidy inducers in myelofibrosis patients. Disclosures: Pardanani: Sanofi-Aventis: Clinical trial support Other; YM BioSciences: Clinical trial support, Clinical trial support Other; Bristol-Myers Squibb: Clinical trial support, Clinical trial support Other.

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