ALBERT

Description: The mechanism of G-CSF-induced hematopoietic progenitor/stem cell (HPC/HSC) mobilization from bone marrow microenvironment to peripheral blood has been related to local production by neutrophils of proteases, including elastase, cathepsin G and metalloproteinases (MMPs). We previously showed that in vitro migration of hematopoietic cells across a layer of human marrow stromal cells (MSC) is induced when stimulated by G-CSF. In the present study, we investigated the role of MMPs produced by MSC used for the trans-stromal migration of MO7e line and marrow CD34+ cells. Normal human MSC were previously grown to confluence on Transwell® filters (pore diameter 5-μm) and then stimulated or not by IL-1 (15 U/mL/Day) or G-CSF (15 ng/mL/Day) for three consecutive days. In a second step, MO7e or marrow CD34+ cells, put in the upper chamber, were allowed to migrate through the layers (4 hrs, SDF-1 100 ng/mL in the bottom chamber). The contribution of MMPs was evaluated in this in vitro trans-stromal migration assay using specific blocking monoclonal antibodies (mAb), anti-MMP-1, anti-MMP-2 and anti-MMP-9 (each at 5 μg/mL). The amounts of antigenic MMP-2 and MMP-9 produced by MSC were evaluated in cell supernatants by enzyme-linked immunosorbent assay (ELISA) and gelatinolytic activity of MMPs by zymography. mRNA expression of MMPs in MSC was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). None of the MSC layers used contained detectable hematopoietic cells since they were all CD45-. Migration of MO7e cells across unstimulated or G-CSF-stimulated stromal cells was significantly inhibited by specific mAb. Percentages of inhibition were 25+/−5 % and 54+/−9 % for anti-MMP-2 mAb, and 18+/−6 % and 53+/−9 % for anti-MMP-9 mAb, respectively. Using the combination of anti MMP-2 and anti-MMP-9, inhibition reached 71+/−24 % across stimulated layers. In contrast, anti-MMP-1 mAb did not show any significant inhibition. Migration of CD34+ across G-CSF-stimulated stromal cells was similarly inhibited with percentages of 38+/−18 % for anti-MMP- 2 mAb, and 34+/−11 % for anti-MMP-9 mAb, while migration was not inhibited again, using anti-MMP-1 mAb. In parallel, we demonstrated that MSC did express G-CSF receptor by RT-PCR. Production of MMPs by unstimulated and G-CSF-stimulated MSC was detected by ELISA, zymography and RT-PCR for MMP-2, and by zymography and RT-PCR for MMP-9. In conclusion, G-CSF-induction of HPC trans-stromal migration involves MMP-2 and MMP-9 but not MMP-1. These findings suggest that microenvironment, in the absence of neutrophils, could have a significant role in G-CSF mobilization of HPC/HSC. A local production of MMP-2 and MMP-9 by marrow stromal cells could be critical in the egress of HSC out of the hematopoietic niche.

Description: Abstract 2264 The alpha4 beta1 integrin (VLA-4) exert a critical role on hematopoiesis by confining hematopoietic stem cells (HSC) and progenitor cells (HPC) within the niche. Previous preclinical studies have pointed out this role showing that HPCs can be mobilized by in vivo administration of a blocking anti-VLA-4 antibody (Ab) (Papayannopoulou et al, 1993). Very recently, two papers (Bonig et al, 2008; Zohren et al, 2008) have shown that such Ab exhibits a similar effect in humans for one month after treatment by natalizumab. In the present study, we have investigated long-term hematopoietic effects (up to 23 months) of repeated infusions of natalizumab in patients treated for multiple sclerosis (n=22). Seven patients have been explored sequentially (every month for one year) and 15 have been studied punctually (6 before and 9 after one year of treatment). We found that peripheral blood leukocytosis was consistently increased for one year in relation to lymphocytes, particularly to B lineage (by 3-fold). In parallel, an increase of circulating HPCs (CD34+ cells and total CFU) was observed and appeared more pronounced with levels above baseline values in all the patients studied (by 6-fold) while no increase of circulating mesenchymal stromal cells (CFU-F) was found. The increase was noted for both lymphoid (T and B lineages) and myeloid (granulo-monocyte, erythroid, and megakaryocyte) committed progenitor cells but also for primitive HPC (CD34+CD38- cells and CAFC). This effect was still found at long-term (up to two years of treatment) for both committed and primitive HPC. In conclusion, the HPC mobilizing effect of chronic administration of anti-VLA-4 Ab in humans involves all types of HPCs (lymphoid and myeloid, committed and primitive ones) and is not exhausted with time. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Bone marrow niche of acute myeloid leukemia (AML) is a highly specialized microenvironment that regulates leukemic progression by favoring leukemic cell quiescence and chemoresistance. Niche components, especially mesenchymal stromal/stem cells (MSCs), have pro-survival effects on leukemia cells by protecting them from chemotherapy-induced apoptosis. The protection mechanisms of AML cells by the microenvironment are extensively studied to identify pharmacological targets to prevent AML relapse after chemotherapy. Direct intercellular communications between hematopoietic stem cells and MSCs involve connexins, such as connexin 43 (Taniguchi Ishikawa E et al. Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells. Proc Natl Acad Sci U S A. 2012,109:9071-6). The connexins are key components of gap junctions and we postulated in this study that blocking their assembly could modify cell-cell interactions in the leukemic niche and consequently the chemoresistance. To this end we evaluated the effects of carbenoxolone (CBX), a glycyrrhetinic acid derivative known to block gap junctions (Rozental R et al. How to close a gap junction channel. Efficacies and potencies of uncoupling agents. Methods Mol Biol 2001;154:447-76), already evaluated in the treatment of human diseases. Methods Primary AML blasts isolated from bone marrow patients and 8 AML cell lines (KG1-a, KG1, HL-60, THP-1, MOLM-13, MV4-11, ML2, NB4) were exposed to CBX to assess cell viability and proliferation (Trypan blue exclusion and MTT assays). To study the combined drug effects of CBX and cytarabine (Ara-C), isobolograms were determined by co-treatment of AML cell lines with various doses of these two molecules. The metabolic effects of CBX on AML live cells were investigated with Seahorse® analyzer to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) and high-throughput metabolic profiling was performed by OmniLog® technology. Direct cell-cell interactions were studied in a co-culture system of AML cells and bone marrow primary MSCs combining or not CBX (150 µM) and Ara-C (1 µM). After 48h (37°C, 5% CO2), AML cells adherent to MSCs were collected and the two populations were discriminated by flow cytometry studies (AML cells: CD45+CD90- / MSCs: CD45-CD90+) to quantify cell number apoptosis/necrosis (AnnexinV/7AAD assay). Gap junction inhibition by CBX was checked by calcein transfer (dye transferred mainly by gap junction channels) from labeled MSCs to AML cells in the co-culture system. Statistical analyses were performed with Mann-Whitney-Wilcoxon test. Results Treatment of AML cell lines with CBX reduced cell growth and viability in a time- and dose-dependent manner and the CBX IC50 was around 150 µM. Moreover, the exposure for 48h of primary AML blast to this dose of CBX induced a two time decrease in the number of viable cells. Interestingly, the isobolograms of the 8 AML cells lines identified three different profiles of resistance to chemotherapy and a synergistic effect between CBX and Ara-C. Regarding the energy metabolism, the analysis of all AML cell lines showed a major reduction of OCR and ECAR after treatment with CBX (150 µM, 48h) regardless their chemoresistance to Ara-C. Moreover, treated AML cells lacked their metabolic capacities for utilization of numerous substrates. In the co-culture experiments, contact with MSCs induced resistance to Ara-C; the apoptosis/necrosis rate observed after Ara-C exposure was 4.3- and 6.4-times decreased by MSC-contact for KG1a cells (n=5, p=0.0115) and primary blasts (n=5, p=0.0001), respectively. Co-treatment of leukemic cells with CBX and Ara-C reversed these deleterious effects of MSC-contact; the apoptosis/necrosis rate observed in MSC-contact leukemic cells was 5- and 2-times increased by CBX co-treatment for KG1a cells (n=5, p=0.007) and primary blasts (n=5, p=0.042), respectively. Finally, CBX induced a 48%-decrease in calcein transfer between leukemic cells and MSCs, highlighting its role as a gap junction inhibitor. Conclusion Niche-induced chemoresistance is associated with the mechanism of AML relapse after initial well-conducted chemotherapy. Combined to chemotherapy, CBX could be of interest to reduce the deleterious effects of leukemic niche by targeting gap junctions. Disclosures No relevant conflicts of interest to declare.

Description: 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.

Description: Abstract 1058 Redox metabolism plays an important role in self-renewal and differentiation of hematopoietic cells and it has been recently established by the Guy Sauvageau's group (Institute for Research in Immunology and Cancer, Montréal, QC) that glutathione peroxydase 3 (GPx-3) promotes competitiveness of Hoxa9-Meis1 induced leukemic stem cells in which Gpx3 overexpression is concomitant of a decrease in H2O2 level and inactivation of p38 MAPK (Herault O et al, ASH annual meeting, 2010 - submitted). Leukemic cells located in the bone marrow (BM) are interacting with a microenvironment which plays a crucial role in the development and progression of leukemia. Mesenchymal stromal cells (MSCs) constitute a population of multipotential cells giving rise to the different hematopoietic microenvironmental cells (adipocytes, osteoblasts, chondrocytes, and vascular-smooth muscle-like hematopoietic supportive stromal cells). The aim of our study was to evaluate the effects of MSC-contact on the GPx-3/H2O2/p38 MAPK axis in human leukemic cells and to assess the cell cycle changes associated with the modification of H2O2 metabolism. BM MSCs were obtained from informed and consenting patients undergoing orthopedic surgery, following a procedure approved by the local ethical committee. Nucleated cells harvested from the iliac crest were seeded (5.104 cells per cm2) in α-MEM supplemented with 10% fetal calf serum (FCS), 20 mmol/l L-glutamine, and 100 units/mL penicillin G. Cells were incubated at 37°C in 95% humidified air and 5% CO2. When fully confluent, the layer of adherent cells was trypsinized (0.25% trypsin-EDTA), and the cells were resuspended in culture medium and seeded at 103 cells per cm2 (passage 1 - P1). BM MSCs were used at P2 in all experiments. The three-lineage mesenchymal differentiation of the BM MSCs used was systematically checked by culturing cells in adipogenic, chondrogenic, and osteogenic induction media as previously described (Delorme et al, Blood 2008, 111:2631). The human KG1a leukemic cell line (FAB M0/M1 CD34+ leukemic cells) was cultured in RPMI 1640 with 20 mmoL/L L-glutamine supplemented with 10% FCS, 100 units/mL penicillin G, and 100 mg/mL streptomycin. KG1a cells were seeded at 1.5 105 cells/cm2 and cocultured with MSCs for 72 h. We have defined two distinct localizations of leukemic cells relative to MSC layer: those in supernatant (non-adherent cells) and cells adhering on the surface of MSCs. The expression of antioxydative enzymes, H2O2 level, p38 MAPK activation (T180/Y182), cell cycle, proliferation and immunophenotype of these two cell fractions were evaluated at day 0 and day 3. The expression of SOD1, SOD2, SOD3, CAT, TXN, TXN2, GLRX, GLRX2, GLRX3, GLRX5, PRDX, PRDX2, PDRX3, PRDX4, PRDX5, PRDX6, GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7 and GSR antioxydative genes and CDKN1A (p21CIP1) gene was quantified by qRT-PCR (Universal ProbeLibrary, Roche). SDS-PAGE and western-blot experiments were realized to quantify GPx-3 expression and p38 MAPK activation. Flow cytometry studies were performed: (a) to quantify H2O2 level by dichlorodihydrofluorescein diacetate (DCF-DA) staining; (b) to analyze the cell cycle by staining with 7-aminoactinomycin D (7AAD), Alexa Fluor®488-conjugated anti-human Ki67 and Alexa Fluor®488-conjugated anti-phospho-histone H3 (ser10) antibodies; (c) to track the cell divisions with carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Supernatant of MSCs did not modify the GPx-3/H2O2/p38 MAPK axis in KG1a cells. Conversely, when compared with cells in the supernatant of MSCs, adhering KG1a cells were characterized by the exclusive overexpression of GPX3 antioxydative gene, the induction of GPx-3 production, a major decrease in H2O2 concentration and the inactivation of p38 MAPK. These effects were concomitant of cell cycle inhibition: increase in G0 phase, decrease in S and M phase, overexpression of CDKN1A and reduced mitotic activity (CFSE). Altogether these findings suggest that the bone marrow microenvironment plays a key role in the regulation of the oxidative metabolism of leukemic cells by promoting the inhibition of the H2O2/p38 MAPK axis via the induction of GPx-3. Modulation of the GPx-3/H2O2/p38 MAPK pathway by targeting of microenvironmental interactions in leukemia may have clinical relevance and it will be important to verify if these results can be extended in vivo to other models of human leukemias. Disclosures: No relevant conflicts of interest to declare.

Description: The bone marrow (BM) hematopoietic stem cell (HSC) niche is a specialized structure of the microenvironment, which supports survival and regulates HSC function (i.e. the HSC control of the self-renewal/differentiation balance and migration). The supportive cells involved in the HSC niche are usually named as “stromal cells” but their precise nature remains a matter of debate (in particular, to know whether these cells belong to osteoblastic or to vascular smooth muscle lineage). Mesenchymal stem cells (MSCs) that are present into the BM are characterized by a broad differentiation potential including adipocytic (A), osteoblastic (O) and vascular smooth muscle (V) pathways. Although MSCs are believed to be at the origin of stromal cells, their real function within the niche is unknown. The aim of this study was to investigate in vitro the hematopoietic function (HSC support and migration) of cultured adult BM MSCs non-differentiated and during induced differentiation along A, O and V lineages. MSCs were obtained from BM nucleated cells of patients undergoing orthopedic surgery by culture in expansion medium (alpha-MEM medium with 10% FCS and 1 ng/mL FGF-2). The MSCs were tested before (cultured in expansion medium) and during differentiation induction in appropriate medium for A, O or V lineages (from 3 to 21 days). Interestingly, non-differentiated MSCs already co-expressed O (PTH-receptor), A (leptin) and V (ASMA) markers as assessed by Western blotting. Capacity of MSCs to support hematopoiesis was evaluated by long-term cultures (for 5 wks) with BM CD34+ cells in limiting dilution (CAFC assay), and capacity to control CD34+ cell migration by using Transwells seeded with MSCs (trans-stromal migration assay). We showed that non-differentiated MSCs have the most important capacity to support hematopoiesis (5-week CAFC frequency) and that this capacity was quickly and dramatically lost from 3 days of differentiation towards A (36±2% of non-differentiated values), O (40±3%) and V (38±1%) lineages. This capacity was almost abolished after 14 days of A, O and V differentiation (