scholarly journals Bone Marrow Mesenchymal Stromal Cells and Inflammation Contribute to ETV6-RUNX1+ Preleukemic Cells Persistence and DNA Damaging

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3918-3918
Author(s):  
Linda Beneforti ◽  
Erica Dander ◽  
Silvia Bresolin ◽  
Clara Bueno ◽  
Geertruy te Kronnie ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Inflammatory Cytokines ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Fold Increase ◽  
Mrna Levels ◽  
Soluble Factors ◽  
Cd34 Cells

Abstract INTRODUCTION ETV6-RUNX1 (ER), generated from translocation t(12;21), is the most frequent fusion gene in pediatric cancers, exclusively leading to B-Cell Precursor Acute Lymphoblastic Leukemia. Translocation occurs in fetal hematopoietic stem-progenitor cells (HSPC) but it is insufficient for disease. ER, in fact, is an aberrant transcription factor that expands a silent preleukemic clone with enhanced self-renewal and partial B cell differentiation. Secondary hits are thus required to complete transformation. Epidemiological and experimental data indicate that infections/inflammation play an important role in the preleukemia to leukemia transition. We previously demonstrate that TGFβ1, a pleiotropic cytokine produced after inflammation, favored the persistence of ER+Ba/F3 cells and selected putative preleukemic stem cells in ER+umbilical cord blood (UCB) CD34+cells. We also demonstrated that ER+Ba/F3 showed altered expression of adhesion molecules and impaired migration towards CXCL12. Migration, physical interactions and response to soluble factors determine HSPC fate in the Bone Marrow (BM) niche. BM Mesenchymal Stromal Cells (MSC) are non-redundant regulators of HSPC in the niche; in addition, they possess pro- and anti-inflammatory properties, representing a bridge between hemopoiesis and inflammation. Finally, dysfunctions in MSC can induce myelodisplasia and secondary myeloid leukemia, while MSC inflammation cause genotoxicity in HSPC predicting myeloid leukemia evolution in predisposing syndromes. On that basis, we questioned if interaction between ER+cells, MSC and inflammation could favor preleukemic clone persistence and progression. METHODS The murine proB cell line Ba/F3 was transfected to generate an inducible ER-V5tag expressing model (Ford A, Palmi C, 2009). BM-MSC were characterized and cultured for controlled passages. UCB-CD34+cells were immunomagnetically isolated and lentivirally transduced with pRRL-eGVP or pRRL-ER-eGFP constructs. Cells were treated with IL6/IL1β/TNFα inflammatory cytokines. RESULTS Gene Expression Profile shows that ER affects pathways involved in inflammatory response, cell cycle, apoptosis and migration in Ba/F3. In particular, ER+ cells overexpress CXCR2, a chemokine receptor also implicated in cancer, (MFI: ER=1378±807 vs ctr=284±167, p<0.05) and highly migrate toward its ligand CXCL1 (% migrated cell/input: ER=21.5±6.7 vs ctr=2.2±1.8, p<0.01). Interestingly, MSC increases CXCL1 secretion after inflammatory stimulation (murine MSC, pg/mL: basal=78±28 vs +infl.ck=30162±4760, p<0.01). In accordance, ER+ Ba/F3 are highly attracted by inflamed MSC supernatants (% migrated cell/input: ER=30.2±9.1 vs ctr=14.3±9.6, p<0.01) in a CXCR2-dependent manner. Coculturing control and ER+ Ba/F3 with MSC and inflammatory cytokines favored the persistence of preleukemic cells in the coculture (% ER+ fold increase: +MSC vs +MSC+infl.ck = 2.62±0.94, p<0.01). The effect is mediated by soluble factors and results from decreased survival in control (% ann-V negative cells: +MSC=68.4±5.7 vs +MSC+infl.ck=48.2±1.3, p<0.05) but not ER+ Ba/F3; cell proliferation was reduced in both, but the effect was stronger on control Ba/F3 (CSFE MFI fold increase +MSC vs +MSC+infl.ck: ER=2.2±0.6, p<0.001; ctr=4.4±1.8, p<0.05). However, CXCL1 is not implicated. Phosphorilation of histone H2AX and AID mRNA levels, which are basally higher in ER+ Ba/F3, further increase in both normal and ER+ Ba/F3 cocultured with MSC and inflammatory cytokines, confirming the genotoxicity of MSC inflammation (γH2AX MFI fold increase +MSC vs +MSC+infl.ck: ER=2.5±1, p<0.05; ctr=2.8±1.2, p<0.01) (AID mRNA fold increase basal vs +MSC+infl.ck: ER=6.3±1.6, p<0.05; ctr=14.6±11). Finally, preliminary data show a higher migration towards inflamed MSC also in ER+ UCB-CD34+cells (% migrated cell/input: ER=21.2±2.4 vs ctr=5.2±0.6, p<0.01). CONCLUSIONS ER expression increases migration towards inflamed BM-MSC supernatants in murine proB cells. Interestingly, MSC and inflammation create favoring microenvironmental conditions for preleukemic cells persistence and DNA damage accumulation. Preliminary results show that inflamed MSC highly attract human ER-expressing UCB-CD34+as well. Collectively, our data support the importance of ER-driven alterations in hematopoietic/BM stromal cells interactions in the leukemogenic process. Disclosures No relevant conflicts of interest to declare.

scholarly journals Characterization of the Hematopoietic Supporting Activity of Osteoblasts Derived from Bone Marrow Mesenchymal Stromal Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4358-4358
Author(s):  
Manal Alsheikh ◽  
Roya Pasha ◽  
Nicolas Pineault
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Soluble Factors ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
The Impact ◽  
Myeloid Progenitors

Abstract Osteoblasts (OST) found within the endosteal niche are important regulators of Hematopoietic Stem and Progenitor Cells (HSPC) under steady state and during hematopoietic reconstitution. OST are derived from mesenchymal stromal cells (MSC) following osteogenic differentiation. MSC and OST secrete a wide array of soluble factors that sustain hematopoiesis. Recently, we showed that media conditioned with OST derived from MSC (referred as M-OST) after 6 days of osteogenic differentiation were superior to MSC conditioned media (CM) for the expansion of cord blood (CB) progenitors, and CB cells expanded with M-OST CM supported a more robust engraftment of platelets in NSG mice after transplantation. These findings raised the possibility that M-OST could be superior to MSC for the ex vivoexpansion HSPC. In this study, we set out to test the hypothesis that the growth modulatory activity of M-OST would vary as a function of their maturation status. The objectives were to first monitor the impact of M-OST differentiation and maturation status on the expression of soluble factors that promote HSPC expansion and in second, to investigate the capacity of M-OST CMs prepared from M-OST at distinct stages of differentiation to support the expansion and differentiation of HSPCs in culture. M-OST at distinct stages of differentiation were derived by culturing bone marrow MSC in osteogenic medium for various length of time (3 to 21 days). All CB CD34+ enriched (92±7% purity) cell cultures were done with serum free media conditioned or not with MSC or M-OST and supplemented with cytokines SCF, TPO and FL. We first confirmed the progressive differentiation and maturation of M-OST as a function of osteogenic culture length, which was evident by the induction of the osteogenic transcription factors Osterix, Msx2 and Runx2 mRNAs, the gradual increase in osteopontin and alkaline phosphatase positive cells and quantitative increases in calcium deposit. Next, we investigated the expression in MSC and M-OSTs of genes known to collaborate for the expansion of HSPCs by Q-PCR. Transcript copy numbers for IGFBP-2 increased swiftly during osteogenic differentiation, peaking at day-3 (˃100-fold vs MSC, n=2) and returning below MSC level by day-21. In contrast, ANGPTL members (ANGPTL-1, -2, -3 and -5) remained superior in M-OSTs throughout osteogenic differentiation with expression levels peaking around day 6 (n=2). Next, we tested the capacity of media conditioned with primitive (day-3, -6), semi-mature (day-10, -14) and mature M-OST (day-21) to support the growth of CB cells. All M-OST CMs increased (p˂0.03) the growth of total nucleated cells (TNC) after 6 days of culture compared to non-conditioned medium used as control (mean 2.0-fold, n=4). Moreover, there was a positive correlation between cell growth and M-OST maturation status though differences between the different M-OST CMs tested were not significant. The capacity of M-OST CMs to increase (mean 2-fold, n=4) the expansion of CD34+ cells was also shared by all M-OST CMs (p˂0.05), as supported by significant increases with immature day-3 (mean ± SD of 18 ± 6, p˂0.02) and mature day-21 M-OST CMs (14 ± 5, p˂0.05) vs. control (8 ± 3, n=4). Conversely, expansions of TNC and CD34+ cells in MSC CM cultures were in-between that of control and M-OST CMs cultures. Interestingly, M-OST CMs also modulated the expansion of the HSPC compartment. Indeed, while the expansion of multipotent progenitors defined as CD34+CD45RA+ was promoted in control culture (ratio of 4.5 for CD34+CD45RA+/CD34+CD45RA- cells), M-OST CMs supported greater expansion of the more primitive CD34+CD45RA- HSPC subpopulation reducing the ratio to 3.3±0.4 for M-OST cultures (cumulative mean of 10 cultures, n=2). Moreover, the expansions of CD34+CD38- cells and of the long term HSC-enriched subpopulation (CD34+CD38-CD45RA-Thy1+) in M-OST CM cultures were respectively 2.7- and 2.8-fold greater than those measured in control cultures (n=2-4). Finally, the impact of M-OST CMs on the expansion of myeloid progenitors was investigated using a colony forming assay; expansion of myeloid progenitors were superior in all M-OST CM cultures (1.6±0.2 fold, n=2). In conclusion, our results demonstrate that M-OST rapidly acquire the expression of growth factors known to promote HSPC expansion. Moreover, the capacity of M-OST CMs to support the expansion of HSPCs appears to be a property shared by M-OST at various stages of maturation. Disclosures No relevant conflicts of interest to declare.

Alterations in multipotent mesenchymal stromal cells from the bone marrow of acute myeloid leukemia patients at diagnosis and during treatment

2019 ◽  
Vol 60 (8) ◽  
pp. 2042-2049
Author(s):  
Irina N. Shipounova ◽  
Nataliya A. Petinati ◽  
Alexey E. Bigildeev ◽  
Tamara V. Sorokina ◽  
Larisa A. Kuzmina ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Acute Myeloid

scholarly journals Mesenchymal Stromal Cells Orchestrate Follicular Lymphoma Cell Niche Through the CCL2-Dependent Recruitment and Polarization of Monocytes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1566-1566
Author(s):  
Fabien Guilloton ◽  
Gersende Caron ◽  
Cédric Ménard ◽  
Céline Pangault ◽  
Patricia Amé-Thomas ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Cell Growth ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Gene Set Enrichment Analysis ◽  
Cell Niche

Abstract Abstract 1566 Accumulating evidence indicates that infiltrating stromal cells contribute directly and indirectly to tumor growth in a wide range of solid cancers and hematological malignancies. In follicular lymphoma (FL), malignant B cells are found admixed with heterogeneous lymphoid-like stromal cells within invaded lymph nodes and bone marrow (BM). In addition, in vitro functional studies have underlined that mesenchymal cells recruit malignant FL B cells and protect them from spontaneous and drug-induced apoptosis. In particular, we have previously demonstrated that mesenchymal stromal cells (MSC) efficiently support in vitro FL B-cell survival, especially after their engagement towards lymphoid differentiation through treatment with TNF-α and Lymphotoxin-α1β2 (TNF/LT) or after coculture with malignant B cells. However, the mechanisms of this supportive activity remain largely unknown. In this study, we used Affymetrix U133 Plus 2.0 microarrays, to compare the gene expression profile (GEP) of bone marrow-derived MSC (BM-MSC) obtained from 10 FL patients at diagnosis versus 6 age-matched healthy donors (HD). In these conditions, neither the CFU-F concentration in the BM nor the cumulative population doubling of BM-MSC significantly differed between HD and FL patients. Unsupervised analysis was able to perfectly segregate FL-MSC from HD-MSC and we identified, using supervised analyzes, a list of 408 probesets defining FL-MSC signature, including 320 nonredundant genes upregulated in FL-MSC compared to HD-MSC. We then defined the GEP of human lymphoid-like stroma using HD-MSC treated in vitro by TNF/LT and demonstrated, by a Gene Set Enrichment Analysis (GSEA) approach, that the FL-MSC signature is significantly enriched for genes associated with a lymphoid-like commitment. Interestingly, CCL2 was strongly overexpressed by FL-MSC, was upregulated in HD-MSC by coculture with malignant B cells, and was detected at a higher level in FL BM plasma compared to normal BM plasma (504.4 pg/mL [23.8-4413] versus 33.9 pg/mL [5-126.1]; P <.01). In agreement, FL-MSC triggered a more potent CCL2-dependent monocyte migration than HD-MSC. Moreover, FL-MSC and macrophages cooperated to sustain malignant B-cell growth through both protection from apoptosis and enhancement of cell proliferation. Finally, FL-MSC promoted monocyte differentiation towards a proangiogenic LPS-unresponsive phenotype close to that of tumor-associated macrophages. We unraveled a key role for the Notch pathway in this process and identified an overexpression of JAGGED1 in FL-MSC compared to HD-MSC. Altogether, these results highlight the complex role of FL stromal cells that promote direct tumor B-cell growth and orchestrate FL cell niche. The identification and characterization of this intricate network of cell interactions may provide novel therapeutic targets in this disease. Disclosures: No relevant conflicts of interest to declare.

Humoral Effect of a B-Cell Tumor on the Bone Marrow Multipotent Mesenchymal Stromal Cells

2021 ◽  
Vol 86 (2) ◽  
pp. 207-216
Author(s):  
Nataliya A. Petinati ◽  
Alexey E. Bigildeev ◽  
Dmitriy S. Karpenko ◽  
Natalia V. Sats ◽  
Nikolay M. Kapranov ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Cell Tumor

IL-7 Effects on Thymocyte Progenitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3318-3318
Author(s):  
Nahed El Kassar ◽  
Baishakhi Choudhury ◽  
Francis Flomerfelt ◽  
Philip J. Lucas ◽  
Veena Kapoor ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cell ◽  
Notch Signaling ◽  
Stromal Cells ◽  
T Cell Development ◽  
Fold Increase ◽  
Cell Development ◽  
B Cell Development ◽  
Over Expression

Abstract IL-7 is a non-redundant cytokine in T cell development. We studied the role of IL-7 in early T-cell development using a model of transgenic (Tg) mice with the murine IL-7 gene under control of the lck proximal promoter. At high IL-7 over-expression (x39 fold increase at day 1 in total thymic tissue), we observed a disruption of TCRαβ development along with increased B cell development in the thymus (7- to 13-fold increase) (El Kassar, Blood, 2004). In order to further explore abnormal T and B cell thymic development in these mice, we first confirmed that they both arise in parallel and were non-cell autonomous, by in vivo injection of neutralizing anti-IL-7 MAb and mixed bone marrow chimera experiments. Using a six color flow cytometry analysis, we found a dramatic decrease of the early thymocyte progenitors (ETPs, lin−CD44+CD25−c-kithiIL-7R−/lo) in the adult Tg mice (x4.7 fold decrease). Lin−CD44+CD25−c-kit+ thymocytes were sorted and cultured on OP9 and OP9 delta-like1 (OP9-DL1) stromal cells (kindly provided by Pr Zuniga Pflucker). At day 14, we observed an important decrease of T cell development (54% vs. 1% of DP cells) and an increase of NK cells (x5 fold increase) in the Tg-derived DN1 cell culture. DN2 (Lin−CD44+CD25−c-kit+) Tg thymocytes showed the same, but less dramatic abnormalities. While DN1 progenitors developed effectively into B220+CD19+ cells on OP9 stromal cells, no B cell development was observed on OP-DL stromal cells from DN1-Tg derived progenitors or by addition of increasingly high doses of IL-7 (x10, x40, x160) to normal B6-derived DN1 progenitors. Instead, a block of T-cell development was observed with increased IL-7. We hypothesized a down regulation of Notch signaling by IL-7 over-expression and analyzed by FACS Notch expression in the DN thymocytes. By staining the intra-cellular part of Notch cleaved after Notch 1/Notch ligand activation, Tg-derived DN2 cells showed decreased Notch signaling. More importantly, HES expression was decreased in the DN2, DN3 and DN4 fractions by semi-quantitative PCR. Sorted Pro/Pre B cells from Tg thymi showed TCR Dβ1-Jβ1 rearrangement indicating their T specific origin, in opposition to Pro/Pre B cells sorted from the bone marrow of the same mice. We suggest that more than one immature progenitor seeds the thymus from the bone marrow. While ETPs had T and NK proliferative capacity, another thymic progenitor with B potential may be responsible for thymic B cell development in normal and IL-7 Tg mice. Finally, IL-7 over-expression may induce a decreased Notch signaling in thymic progenitors, inducing a switch of T vs. B lineage development.

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

Blood ◽  
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.

Molecular Characterization of the Leukemic Niche in Chronic Myeloid Leukemia (CML) and Evaluation of a Leukemia / Niche Cross-Talk

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 908-908
Author(s):  
Djamel Aggoune ◽  
Nathalie Sorel ◽  
Sanaa El Marsafy ◽  
Marie Laure Bonnet ◽  
Denis Clay ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mrna Expression ◽  
Expression Pattern ◽  
Myeloid Leukemia ◽  
Gene Expression Pattern ◽  
Fold Increase ◽  
Leukemic Cells ◽  
Cd34 Cells

Abstract Abstract 908 There is growing evidence that the bone marrow microenvironment could participate to the progression of chronic myeloid leukemia (CML). Recent data show indeed that placental growth factor (PGF) expression is highly induced in stromal cells from CML patients although they are not part of the leukemic clone as they are Ph1-negative (Schmidt et al, Cancer Cell 2011). It is possible that leukemic cells instruct the niche components via extracellular or contact signals, transforming progressively the “normal niche” into a functionally “abnormal niche” by inducing aberrant gene expression in these cells, similar to the pattern that has been identified in cancer-associated fibroblasts (CAF). In an effort to identify the differential gene expression pattern in the CML niche, we have undertaken two strategies of gene expression profiling using a Taqman Low Density Arrays (TLDA) protocol designed for 93 genes involved in antioxidant pathways (GPX, PRDX, SOD families), stromal cell biology (Collagen, clusterin, FGF, DHH), stem cell self-renewal (Bmi1, MITF, Sox2) and hematopoietic malignancies (c-Kit, hTERT, Dicer, beta-catenin, FOXO3). The first strategy consisted in the analysis of mesenchymal stem cells (MSCs) isolated from the bone marrow of newly diagnosed CP-CML patients (n=11). As a control, we have used MSCs isolated from the bone marrow of age-matched donors (n=3). MSCs were isolated by culturing 6–8.106 bone marrow mononuclear cells in the presence of b-FGF (1 ng/ml). At 2–3 weeks, cells were characterized by the expression of cell surface markers (CD105+, CD90+) and by their potential of differentiation towards osteoblastic, chondrocytic and adipocytic lineages. The second strategy aimed to study the potential instructive influence of leukemic cells in the gene expression program of normal MSC after co-culture with either the UT7 cell line expressing BCR-ABL (3 days) or with CD34+ cells isolated from CP-CML at diagnosis (5 days) as compared to co-culture with cord blood CD34+ cells. After culture, CD45-negative MSC were cell-sorted and analyzed by TLDA. All results were analyzed using the StatMiner software. Results: TLDA analysis of gene expression pattern of MSC from CML patients (n=11) as compared to normal MSCs (n=3) identified 6 genes significantly over-expressed in CML-MSC: PDPN (10-Fold Increase), V-CAM and MITF (∼8 Fold increase), MET, FOXO3 and BMP-1 (∼ 5 Fold increase). To confirm these results we have performed Q-RT-PCR in a cohort of CML-MSC (n= 14, including the 11 patients as analyzed in TLDA) as compared to normal MSC. High levels of PDPN (Podoplanin, ∼8 fold increase), MITF (Microphtalmia Associated Transcription factor, 4-Fold) and VCAM (Vascular Cell Adhesion Protein, 2 fold increase) mRNA were again observed on CML MSCs. Our second strategy (co-culture of normal MSC with BCR-ABL-expressing UT7) revealed an increase of IL-8 and TNFR mRNA expression in co-cultured MSCs (∼5-fold ) whereas there was a major decrease in the expression of DHH (∼ 25-fold) upon contact with BCR-ABL-expressing cells. No modification of the expression of PDPN, MITF or VCAM was noted in normal MSC after this 3-day co-culture strategy using UT7-BCR-ABL cells. Current experiments are underway to determine if primary CD34+ cells from CML patients at diagnosis could induce a specific gene expression pattern in normal MSC after 5 days of co-culture. PDPN is a glycoprotein involved in cell migration and adhesion, acting downstream of SRC. It has been shown to promote tumor formation and progression in solid tumor models and is highly expressed in CAFs. MITF is a bHLH transcription factor involved in the survival of melanocyte stem cells and metastatic melanoma. Finally, high VCAM1 mRNA expression by MSCs from CML patients could be involved in increased angiogenesis known to be present on CML microenvironment. In conclusion, our results demonstrate an abnormal expression pattern of 3 important genes (PDPN, MITF and VCAM1) in MSC isolated in CP-CML patients at diagnosis. The mechanisms leading to an increased mRNA expression (instructive or not instructive by leukemic cells) and their relevance to CML biology are under evaluation. Our results, confirming previous data, suggest strongly the existence of a molecular cross-talk between leukemic cells and the leukemic niche. The elucidation of such aberrant pathways in the microenvironment could lead to the development of “niche-targeted” therapies in CML. Disclosures: Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.

A Plant Triterpenoid Avicin D Stimulates Adipocytic Differentiation of Bone Marrow Stromal Cells and Promotes Their Pro-Survival Effects On Acute Monoblastic Leukemia Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4315-4315
Author(s):  
Yoko Tabe ◽  
Linhua Jin ◽  
Saiko Kazuno ◽  
Tsutomu Fujimura ◽  
Hiromichi Matsushita ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Cycle Arrest ◽  
Inflammatory Cytokines ◽  
Stromal Cells ◽  
Fold Increase ◽  
Leukemia Cells ◽  
U937 Cells ◽  
Cycle Arrest ◽  
Adipocytic Differentiation

Abstract Abstract 4315 A natural triterpene Avicin D induces apoptosis in various tumor cells and regulates cellular metabolism via Glucocorticoid receptor (GR) signalling, known to be involved in adipocyte differentiation (Tang, Annu Rev Biochem. 2012;81:715). Since adipocytes represent an essential component of the aging bone marrow (BM) microenvironment (Tabe, Blood 2004;103:1815), and promote monocytic differentiation and survival of monoblastic leukemia cells (Tabe, ASH, 2011), we examined the potential of Avicin D to modulate BM adipocytes and its effects on the survival of the leukemic cells. In mature adipocytes derived from BM mesenchymal stem cells (MSCs), Avicin D (1ƒÊM, 48 hrs) significantly increased the number of lipid vesicles without apoptosis induction or significant cell cycle arrest. This was associated with upregulation of adipose-specific genes, PPARG and fatty acid binding protein 4 (FABP4), and inflammation-related haptoglobin (HP) gene (p<0.005, Figure 1). Avicin D further enhanced leptin release from adipocytes and inflammatory cytokines production, IL-6 and IL-8, from both MSCs and adipocytes (Figure 2). Of note, Avicin D increased pro-survival Bcl-2 mRNA expression in adipocytes (fold increase compared to MSCs: MSCs+Avicin D 1.0±0, adipocytes 1.8±0.1, adipocytes+Avicin D 4.4±0.6, p=0.02). We next investigated the effects of MSCs and adipocytes pre-treated with 1mM Avicn D for 24 hours on co-cultured monoblastic leukemia cell line U937. Avicin D (1ƒÊM) moderately (26±5 %) inhibited cell proliferation of U937 cells cultured alone as detected by MTT assay. Serum-starvation induced cell death in U937 cells was inhibited by MCS or adipocyte co-culture, and further blocked by Avicin D-pretreated adipocytes, which was accompanied with cell cycle arrest (sub-G1 fraction; control U937 36.1±7.1%, co-culture with MSC 17.9±4.1%, MSC+Avicin D 20.9±3.9%, adipocyte 16.9±2.9, adipocyte+Avicin D 11.5±0.8%, p=0.03, G0/G1 phase; control 39.1±5.9%, with MSC 44.3±1.0%, MSC+Avicin D 40.3±1.3%, adipocyte 40.6±1.3, adipocyte+Avicin D 50.4±2.3%, p=0.03). Co-culture of U937 cells with Avicin D-pretreated adipocytes induced upregulation of PPARG and CD36, markers of monocytes/macrophages maturation in U937 cells (fold increase compared to control U937: PPARG: co-culture with adipocytes 2.1±0.4, adipocytes+Avicin D 13.8±6.2; CD36: adipocytes 2.2±0.4, adipocytes+Avicin D 4.7±2.9). Co-culture with Avicin D-pretreated adipocytes also caused 3.1 ±0.4 fold increase of BCL2 mRNA in U937 cells compared to the one of co-cultured with control adipocytes (p=0.02). The purity of U937 cells separated from adipocytes was confirmed by lack of CD90 mRNA expression by PCR. Utilization of the proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ) with two-dimensional-liquid chromatography-tandem mass spectrometry allowed the identification of 1,634 proteins. We found changes in 14 proteins in U937 cells co-cultured with Avicin D-pretreated adipocytes compared to untreated adipocytes. Among 13 down-regulated proteins, three are involved in pyruvate metabolism (Glyoxalase I; p=0.01, Lactate dehydrogenase B; p=0.02, Pyruvate kinase; p=0.03), and four proteins participate in cell cycle progression (DNA replication licensing factor MCM4, MCM5; p=0.02, p=0.03, DNA-dependent protein kinase; p=0.02, Interleukin enhancer-binding factor 3; p=0.005). Voltage-dependent anion-selective channel protein 2, which is involved in the mitochondrial apoptotic pathway via regulation of Bcl-2, and an enzymatic antioxidant Superoxide dismutase were up-regulated (p=0.04, p=0.03). These data indicate that U937 cells co-cultured with Avicin D pretreated adipocytes were induced to undergo cell cycle arrest, escape apoptosis with down-regulation of glucose metabolism. Altogether, these results suggest that Avicin D induces adipocytic differentiation of BM-derived stromal cells, promotes production of leptin and inflammatory cytokines, which in turn supports survival of monocytic leukemia cells. These findings suggest a contributory role of the aging “pro-inflammatory” bone marrow microenvironment in the reduced efficacy of cytotoxic chemotherapy which is prevalent in elderly AML patients. Disclosures: No relevant conflicts of interest to declare.

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