ALBERT

Description: Abstract 1452 Poster Board I-475 Stromal cell-derived factor 1 (SDF-1)/CXCR4 axis plays a major role in regulating the interactions between hematopoietic stem and progenitor cells (HPSC) and their stromal microenvironment within the bone marrow. A second SDF-1/CXCL12 receptor, CXCR7 binds SDF-1 with high affinity but little is known about its function in hematopoiesis. In the present study, we demonstrate that the activity of CXCR7 is crucial for proper maintenance of hematopoietic activity on stromal layers. Using quantitative reverse transcription-PCR analysis, we demonstrate that CXCR7 is highly expressed in stromal cells in contrast to hematopoietic cells showing that it functions primarily in the stromal microenvironment compartment. CXCR7 stable expression in UT7 hematopoietic cells fails to support SDF-1 induced migration and signalling but inhibits migration of CXCR4 expressing cells in paracrine manner. Overexpression of CXCR7 in MS-5 stromal cells lead to a reduction of SDF-1 concentration in the supernatants. In addition, supernatants from these cells had substantially lower efficiency in promoting integrin alpha-4 beta-1–mediated adhesion and migration of Mo7e cells to vascular cell adhesion molecule-1 (VCAM-1) and CS-1/fibronectin than their control GFP counterparts. Moreover, human cord blood CD34+ hematopoietic progenitor cells displayed SDF-1–dependent reduced responses in chemotaxis, transendothelial migration, and up-regulation of adhesion to VCAM-1 when supernatants from CXCR7 expressing MS-5 cells were used compared with supernatants from GFP expressing cells. Finally, phenotypical primitive murine cells displayed reduced hematopoietic activity when cultured on CXCR7 expressing MS-5 cells. This reduced hematopoietic activity is partly reverted when recombinant SDF-1 was added to CXCR7 overexpressing MS-5 cells. Taken together, our results indicate that CXCR7-controlled disponibility of SDF-1 expression influences BM cell migration, adhesion and hematopoietic activity and thus behaves as a decoy receptor regulating the SDF-1 level. Disclosures Bouamar: Association pour la Recherche sur le Cancer: Employment; Cancéropôle IDF: Employment.

Description: Abstract 4570 Stromal cell-derived factor 1 (SDF-1)/CXCR4 axis plays key roles in hematopoiesis regulating the interactions between hematopoietic cells and their stromal microenvironment within the bone marrow, their trafficking from the bone marrow to blood, their proliferation and survival. SDF-1/CXCR4 interactions also participate in the development of leukemic blasts in acute myeloid leukaemia (AML) influencing their trafficking, survival and differentiation. We used the xenotransplantation model of nonobese diabetes/severe combined immunodeficiency γcnull (NOG) mice reconstituted with human primary cells from AML patients and 2 small molecule competitive antagonists of CXCR4, AMD3100 and TN140 to better define the role of CXCR4/SDF-1 on the leukemic blast burden within the bone marrow and in extramedullary sites. In a first set of experiments, we investigated whether there was a correlation between CXCR4 expression or function on leukemic blasts and their ability to engraft the bone marrow of NOG mice in 34 patients. Using flow cytometric analyses, we observed that CXCR4 membrane expression was highly variable between patients. This expression did not correlate with engraftment. In addition, SDF-1 responsiveness evaluated by transwell migration only marginally correlated with engraftment. However, in these initial analyses, we observed that the differences between engrafters and nonengrafters were significant if the cut-point migration is set to 20%. Patients with higher chemotactic response to SDF-1 had a significantly increased NOG repopulating ability. In further studies, NOG mice reconstituted with AML cells from 5 different patients were treated with optimal concentration of either AMD3100 or TN140 for 1 week. We observed that CXCR4 inhibition by TN140 (used as a single therapy) had profound inhibitory effects on the proliferation and the development of extramedullary dissemination of the disease in this xenotransplantation. This patient had FAB M1 leukemia and initially exhibits high CXCR4 level on blast. Our study demonstrated that CXCR4 inhibition in selected patients might be a potent therapy against leukemic development. Disclosures: Bouamar: Association pour la Recherche sur le Cancer: Employment; Cancéropôle IDF: Employment.

Description: Abstract 1440 Poster Board I-463 Nitric oxide (NO) is a small gaseous molecule with diverse roles including the regulation of cell proliferation, differentiation, apoptosis, adhesion and migration. NO is derived from L-arginine by the nitric oxide synthase (NOS) family of enzymes. At least three distinct NOS isoforms have been identified in mammalian cells including the endothelial (eNOS), neuronal (nNOS) and inducible (iNOS). Recently, we have shown that NO donors induce CXCR4 expression in human CD34 positive cells suggesting that NO production may regulate the migration and adhesion of hematopoietic progenitors. To determine the in vivo relevance of these findings and define the role of NO in the biology of hematopoietic progenitor cells, we first investigated NOS expression in hematopoietic and non-hematopoietic cells. Using quantitative reverse transcription-PCR analysis, we demonstrate that nNOS, and eNOS isoforms are highly expressed in Human Umbilical Vein Endothelial Cells (HUVEC) and osteoblastic cell lines, but there was no or low expression of NOS in hematopoietic cells including cell lines and mature blood cells except macrophages that express high level of iNOS. In agreement with RNA analysis, we found large amounts of nitrite (a stable derivative of NO) in the culture medium of stromal (MS-5) and osteoblasic (MG-63) cell lines. To determine the effects of NO on hematopoietic progenitor cells survival, cord blood CD34+ were cultured on MS-5 cells in the presence/absence of NOS inhibitor (L-NAME) for three days, then hematopoietic progenitor numbers were determined by culture in a semi-solid medium and colonies were quantified 14 days later. Treatment with L-NAME reduced colony number by 33, 42 and 54% with 10, 100 and 500 μM L-NAME concentrations, respectively. This effect is NO specific since the diminution of progenitor number was reverted by adding NO donor in the culture medium. These results indicate that NO is produced by bone marrow stromal cells. This production regulates the survival of hematopoietic progenitors in vitro. Further experiments are needed to determine if these effects are dependent on the regulation of CXCR4/SDF-1 signaling. Disclosures Jouni: Région IDF: Employment. Bouamar: Association pour la Recherche sur le Cancer: Employment; Cancéropôle IDF: Employment.

Description: Abstract 1308 Megakaryocytes (MK) are unique mammalian cells that undergo polyploidization during differentiation, which leads to an increase in cell size and protein production that precedes platelet production. The molecular basis of MK polyploidization, denoted endomitosis, which is strongly altered in hematological malignancies and various other disease states, remain poorly understood. Recent evidence demonstrates that endomitosis is a consequence of a late failure in cytokinesis, associated with a contractile ring defect. Here, we demonstrate that the myosin non-muscle IIB heavy chain (MYH10) is expressed in immature human and mouse megakaryocytes and its expression is repressed during differentiation. In immature MK, MYH10 is specifically localized in the contractile ring while MYH9 is mainly present in the cytoplasm suggesting that they occupy two different functions during MK differentiation. Importantly, MYH10 down-modulation by shRNA or by addition of a chemical inhibitor blebbistatin, increases polyploidization by inhibiting the return of 4N cells to 2N. Conversely, re-expression of MYH10 in MKs prevents polyploidization and the transition of 2N cells to 4N cells. Furthermore, we demonstrated that RUNX1 directly repress the transcription of MYH10. In vitro and in vivo RUNX1 invalidation inhibits MK polyploidization and increases expression of MYH10. Accordingly, in patients with a germline mutation of RUNX1 (FPD/AML), MYH10 is still expressed in platelets. Altogether ours results demonstrated that the RUNX1-mediated silencing of MYH10 is needed for the switch from mitosis to endomitosis linking thus polyploidization with MK differentiation. Disclosures: No relevant conflicts of interest to declare.