ALBERT

Description: Abstract 1804 Introduction: Phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is a fundamental pathway for the regulation of cell proliferation, survival, adhesion, migration, and metabolism in a variety of physiological and pathological processes. The pathway and its downstream effectors are frequently activated in patients with acute leukemias, chronic leukemias, various types of lymphomas, and multiple myeloma (MM). In this study we investigated the expression of the different PI3K catalytic subunits in MM and effect of three different PI3K inhibitors on the interaction of MM cells with the bone marrow (BM) microenvironment. Methods and Results: First we characterized the baseline expression of the different PI3K-p110-alpha, beta, gamma and delta catalytic subunits in MM cell lines (MM1s, OPM1, OPM2, H929, RPMI, INA6, U266, and U266LR7) by immnunobloting. PI3K-p110-alpha was highly expressed in MM1s and RPMI8226; PI3K-p110-beta was highly expressed in all cell lines; PI3K-p110-gamma was highly expressed in OPM1 and OPM2; and PI3K-p110-delta was highly expressed in MM1s and INA6. Furthermore, we investigated BKM120, a novel pan-PI3K inhibitor (Novartis, Swizerland). MM cells (MM1s, H929, OPM1, and OPM2) were treated with increasing concentrations of BKM120 (0, 100, 250, 500 and 1000 nM) for 4 hrs, labeled with Calcein-AM, and applied to fibronectin adhesion plate, or to 96-well plate pre-coated with stroma. Cells were incubated for 1 hr at 37C, non-adherent cells were washed and MM adhesion was measured by fluorescence-reader. BKM120 reduced the adhesion of all MM cell lines to fibronectin and stromal cells in a dose-dependent manner. Mechanistically, BKM120 decreased the activation of adhesion-related signaling in MM cells induced by co-culture with stroma including pFAK, pSRC pCoffilin and pMyosin light chain, as detected by immunoblotting. Moreover, BKM120 inhibited the downstream signaling of PI3K: p-Akt, p-P70S6, and p-S6R and regulated the survival of MM cells with or without co-culture with Bone Marrow stroma (IC50- 1uM 48hrs) and caused cell cycle arrest, as detected by PI staining and analyzed by flow cytometry, and decreased the expression of cyclin D1, p-Rb and increased the expression of P27 and P21, as detected by immunoblotting. Furthermore, we compared the activity of BKM120 to other PI3K inhibitors NVP-BEZ-235, a dual PI3K/mTOR inhibitor (Selleck, Houston, TX); and CAL101, a potent PI3K-p110-delta inhibitor (Selleck, Houston, TX); we examined the effect of different PI3K inhibitors (BKM120 500nM, CAL101 500nM or NVP-BEZ-235 100nM) on adhesion of MM1s cells to fibronectin, and found that the three inhibitors decreased the adhesion. Similarly, down-regulation of the expression of the PI3K catalytic subunits in MM1s, using siRNA decreased the adhesion of MM cells to fibronectin. In addition, we tested the effect of inhibition of PI3K on chemotaxis of MM cells. MM1s cells treated with BKM120 500nM, CAL101 500nM or NVP-BEZ-235 100nM for 4hrs, or with knockdown of PI3K by siRNA were applied to the upper chamber of a Boyden-migration assay, and allowed to migrate towards media with or without SDF1 30nM or conditioned media from MM stroma in the lower chamber for 4hrs. Interestingly, BKM120, NVP-BEZ-235 and the PI3K knock down increased significantly the chemotaxis of MM cells towards SDF1 and BM stromal, while CAL-101 had no effect on chemotaxis. These results were in accord with the effect of the drugs on the surface expression of CXCR4; as both BKM120 and NVP-BEZ-235, but not CAL101, increased the expression of CXCR4 in MM cells. Conclusion: we characterized the basal expression of the different PI3K catalytic subunits in MM cells lines, and showed that BKM120 inhibited PI3K signaling including proliferation and cell cycle in MM cells. BKM120 inhibited MM adhesion; an effect which was observed in NVP-BEZ-235 and CAL101, while only BKM120 and NVP-BEZ-235 increased the chemotaxis and CXCR4 surface expression of MM cells. These findings suggest that BKM120 can be used for regulation of MM cell trafficking in vivo by disrupting adhesion of MM cells to the BM and inducting of mobilization, leading to increased sensitivity to therapeutic agents. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Abstract 453 Introduction: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment, which includes stromal (BMSCs) and endothelial cells (ECs), plays a crucial role in MM pathogenesis and drug resistance. We have previously shown that the chemokine stromal cell-derived factor-1 (SDF-1), its receptor-CXCR4, and GTPases in the downstream signaling of the receptor regulate this interaction. Selectins are adhesion molecules which are involved in the primary interaction of lymphocytes with the endothelium. In this study, we have tested the expression of selectins and their ligands in MM, and explored their role in the interaction with the BM milieu and its potential therapeutic applications. Methods AND RESULTS: Flow cytometry and immunohistochemical (IHC) staining of tissue microarrays revealed that P-selectin glycoprotein ligand-1 (PSGL-1, CD162) was over expressed in MM cells from patients (n=20) and cell lines (MM1s, H929, RPMI, OPM1 and OPM2) compared to normal plasma cells (n=3). Gene expression profiling (GEP) analysis showed that the expression of PSGL-1 was directly correlated with MM stage of progression (normal plasma cells, n=15 〈 MGUS, n=20 〈 smoldering MM, n=23 〈 MM, n=68 p

Description: Abstract 1943 Introduction: Plerixafor (Mozobil®), a potent CXCR4 inhibitor, is approved in combination with G-CSF to mobilize hematopoietic stem cells (HSCs) for autologous transplantation in multiple myeloma (MM) and non Hodgkin's lymphoma (NHL). Another area of investigation consists of exploring whether disruption of the CXCR4 pathway by plerixafor could potentiate the effect of chemotherapy in active disease. This study aimed to establish the maximum tolerated dose (MTD) of plerixafor in combination with bortezomib in patients who have active relapse/refractory MM. This was informed by preclinical studies showing that plerixafor induces de-adhesion of MM cells with sensitization to combination therapy with bortezomib in pre-clinical animal models. Methods: Eligibility criteria include: 1) patients with relapsed or relapsed/refractory MM with any prior lines of therapy including bortezomib, 2) measurable disease, 3) not receiving chemotherapy〉 3weeks, or biological/novel therapy for MM 〉 2 weeks. Patients with active disease received plerixafor at the recommended dose sc on days 1–6 of every cycle. Dose levels include 0.16, 0.24, 0.32, 0.40, and 0.48 mg/kg. Bortezomib was given at the recommended dose twice a week on days 3, 6, 10, and 13 every 21 days. Dose levels include 1.0 and 1.3 mg/m2. Bortezomib was given 60–90 minutes after plerixafor. Patients were assessed after every cycle. Patients who had response or stable disease went on to receive a total of 8 cycles without planned maintenance therapy. 4 dose levels were initially planned at a maximum of 0.24 mg/kg plerixafor. The protocol was then modified to include 3 higher doses of plerixafor, to further evaluate the hypothesis that higher doses may induce better chemosensitization. To examine the in vivo effect of plerixafor and bortezomib on de-adhesion of MM cells and other accessory cells of bone marrow, blood samples were obtained from patients at 0, 2, 4 and 24 hours post-plerixafor injection on days 1 and 3, and time points 0, 2, and 4 hours on days 6, 10 and 13 of cycle 1 and examined for the presence of plasma cells or CD34+ cells using flow cytometry. Results: Thirteen patients have been treated to date, three in each cohort with cohort 5 currently enrolling. The median age is 60, the median lines of prior therapy is 2. All of the patients received prior bortezomib. Three patients were assessed by light chain, two patients had extramedullary disease. The median number of cycles on therapy was 5 (1-8). None of the patients came off study due to toxicity. To date, there have been no dose-limiting toxicities. Overall, the combination is very well tolerated. Grade 3 possibly related toxicities include lymphopenia (30%), hypophosphatemia (15%), anemia (8%), and hyponatremia (8%). No grade 2 or higher neuropathy has been noted in these patients. Twelve patients are evaluable for response, including 1 (8%) complete remission (CR) and 1 (8%) minimal response (MR), with an overall response rate including MR of 2 (16%) in this relapsed/refractory population. In addition, 8 (66%) patients had stable disease (SD), and 2 (18%) had progressive disease (PD). We also examined the number of plasma cells, CD34+ HSCs, and other accessory bone marrow cells (including endothelial progenitor cells and plasmacytoid dendritic cells) in the peripheral blood. Analysis of these samples is ongoing, but preliminary data indicate de-adhesion of plasma cells. Conclusions: : The combination of plerixafor and bortezomib is very well tolerated with minimal neuropathy or other toxicities. The responses observed are encouraging in this relapsed/refractory population. The ability to demonstrate transient de-adhesion of MM cells and accessory cells in vivo indicates that these cells can be separated from their protective stromal environment which may make them more sensitive to chemotherapy. This study was supported by R01CA133799-01, and by Genzyme. Disclosures: Ghobrial: Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 2935 Poster Board II-911 INTRODUCTION: Waldenstrom's macroglobulinemia (WM) is a low-grade lymphoma characterized by the presence of lymphoplasmacytic lymphoma cells in the bone marrow (BM). The BM microenvironment was shown to promote growth and proliferation of WM cells. The Eph receptor family are receptor tyrosine kinases RTKs activated by ephrin which a cell-surface protein, and the interaction between the receptor on one cell and the ligand on other cells promotes the activity of the receptor. Eph receptors are known to control processes such as cell growth, proliferation, migration, and invasion, and their expression level was shown to be elevated in a wide range of solid tumors. However, their role in WM was never explored. METHODS AND RESULTS: Using phosphor-RTK array kit, we have screened the activity of 42 RTKs in CD19+ cells from 4 WM patients, WM cell line BCWM1, IgM secreting cell lines MEC1 and RL, and CD19+ cells from the BM of 4 healthy donors. We found that one of the most significant RTKs which showed high activation in all WM cells from patient sample and cells, compared to all normal cells was Eph-B2 receptor. We have used ephrin-B2, the ligand of the receptor Eph-B2, to test the signaling pathways involved in the activity of this receptor. We found, by immunoblotting, that ephrin-B2 induce phosphorylation of the receptor in a bell-curve manner, with the activity peaking in 100 ng/ml, while the expression of the total form of the receptor was unchanged over arrange of 0-1000 ng/ml of ephrin. A comparable activation was found for several cell-adhesion related proteins including FAK, SRC, paxillin, P130 and cofillin. These finding indicated a major role of the Eph-B2 receptor cell adhesion of WM cell. We have coated plates with increasing concentration of ephrin-B2 and tested the adhesion of BCWM1, MEC1 and RL to ephrin. Again, we found the adhesion peaked at the concentration of 100nM of ephrin. In contrast, ephrin-B2 had no chemotactic effect on WM cells. BM microenvironment components including BM stromal cells (BMSCs) and endothelial cells were shown to enhance the proliferation of WM cells detected by thymidine uptake assay. Ephrin-B2 was shown to be expressed on both endothelial cells and BMSCs. The inhibition of either the ephrin-B2 on endothelial cells and BMSCs, or inhibition of the Eph-B2 receptor on WM cell reduced the adhesion of WM cell to both endothelial cells and BMSCs and decreased the increase of WM cell proliferation induced by endothelial cells and BMSCs. The combination of both inhibition ephrin-B2 and Eph-B2 did not have an additive effect compared to each of them alone. Moreover, the inhibition of either ephrin-B2 or Eph-B2 reduced the activation of cell adhesion-related proteins. CONCLUSION: In conclusion, we showed that the Eph-B2/ephrin-B2 axis was activated in WM cells and that it is important for the adhesion and proliferation of WM cells induced by the BM microenvironment. These findings provide a novel therapeutic target for WM. Disclosures: Ghobrial: Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Description: Abstract 471 Multiple myeloma (MM) is characterized by widespread dissemination of the MM cells at diagnosis associated with multiple focal bone lesions, implying (re)circulation of MM cells into the peripheral blood and (re)entrance or homing into new sites of the BM. However, the driving force for MM cells to leave the BM, egress, and home to new BM niches is still not well understood. Hypoxia (low oxygen) in solid tumors was shown to promote metastasis in solid tumors through activation of proteins involved in the endothelial to mesenchymal Transition (EMT). In this study, we hypothesized that MM tumor progression induces hypoxic conditions, which in turn activates EMT related proteins and promotes metastasis of MM cells. To test this hypothesis, we examined levels of hypoxia in MM cells at different stages of tumor progression in vivo in two animal models: the first by injecting MM1s cell to SCID mice, and the second by injecting 5T33MM cells to C57BL/KaLwRijHsd mice. Hypoxic markers were examined using flow cytometry and immunohistochemistry. We found that tumor progression induced hypoxia in both the MM cells and the tumor microenvironment. Similarly, hypoxia induced genes (HIF1a, HIF1b, HIF2b, CREBBP, HYOU1, VEGF1, HIF1a-inhibitory protein) were increased in MM patients (n=68) compared to plasma cells from healthy donors (n=14). Using flow cytometry we found that the number of circulating MM cells increased with the progression; however, the correlation was observed in late stages of the progression but not in the early stages. A better direct correlation was achieved with the hypoxic state of the MM cells in the BM. Circulating MM cells were more hypoxic that MM cells in the BM (especially at low tumor burden). Moreover, we found that the level of hypoxia in MM cells in the PB did not correlate with the hypoxia in the BM. Next, we tested the mechanism in which hypoxia induces cell egress. We found that MM cells isolated from MM patients have higher gene expression of EMT inducing proteins (E-cadherin, SNAIL, FOXC2, TGFb1) in parallel to a decrease of expression in E-cadherin, and we confirmed the downregulation of E-cadherin expression in correlation with the increase of hypoxia in MM cell and cells in the BM microenvironment in vivo. Culturing MM cells under hypoxic conditions increased the expression of HIF1a and HIF2a. In parallel, hypoxia induced acquisition of EMT related features including downregulation of E-cadherin, upregulation of SNAIL, and inhibition of GSK3b. In addition, hypoxia decreased the adhesion of MM cells to stromal cells. To complete the metastatic process after egress, MM cells need to home to new sites in the BM. Therefore we investigated the effect of hypoxia on expression of CXCR4, chemotaxis and homing of MM cells to the BM. Using flow cytometry we found a direct correlation between hypoxia and the expression of CXCR4 in MM cells in vivo using the SCID-MM1s model. These results were confirmed in vitro, where hypoxia increased the expression of CXCR4 at protein and mRNA levels in MM cells. Moreover, the expression of CXCR4 in MM cells isolated from the PB was higher than cells isolated from the BM especially at low tumor burden, correlating with higher hypoxic state of the circulating tumor cells. Functionally, hypoxia increased the chemotaxis of MM cells towards SDF1a in vitro and, using in vivo confocal microscopy, it was shown to accelerate the homing of MM cells to the BM in vivo. To demonstrate that the chemotaxis and homing were CXCR4 dependent, we treated the hypoxic MM cells with AMD3100 (a CXCR4 inhibitor) and showed that it inhibited chemotaxis in vitro and homing of MM to the BM in vivo. In conclusion, we demonstrate that tumor progression induces hypoxia in the MM cells and in the BM microenvironment. Hypoxia activates EMT-related machinery in MM cells, decreases expression of E-cadherin and consequently decreased the adhesion of MM cells to the BM, and enhance egress of MM cells to the circulation. In parallel, hypoxia increases the expression of CXCR4, and consequently increased the migration and homing of MM cells in from the peripheral blood to the BM. Further studies to manipulate hypoxia in order to regulate tumor dissemination as a therapeutic strategy are warranted. Disclosures: Roccaro: Roche: . Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Abstract 142 Introduction: Receptor tyrosine kinases (RTKs) are key regulators of the development and progression of many types of cancer. The Eph receptors represent the largest family of RTKs, which are used extensively throughout embryogenesis and development but detected in adult normal tissues. Elevated levels of expression of Eph receptors was found in a wide range of cancers and correlated with aggressive disease and metastasis. This is, triggered by a wide array of cellular responses, including cell boundary formation, motility, adhesion, and repulsion. Waldenstrom macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow, indicating homing and adhesion of the malignant cells to specific niches in the bone marrow, which provides a protective environment for the survival and proliferation of these cells. Direct adhesion to endothelial cells and several growth factors and cytokines secreted by endothelial cells in the BM were shown to regulate tumor proliferation. In this study we aimed to characterize the role of RTKs, specifically Eph-B2 receptor, in the interaction of WM cells with the BM microenvironment including cell adhesion, proliferation, and cell-cycle. Methods and Results: We first examined the expression and activation (phosphorylation) of different RTKs in primary WM samples and cell lines using an antibody-based RTK-array. Eph-B2 receptor was activated in all patient samples compared to control, with a 5-fold increase in CD19+ WM cells, and we further confirmed that Eph-B2 was phosphorylated in WM cell lines, BCWM.1, and in IgM secreting cell lines RL, MEC-1. We next examined the activity of Ephrin-B2/Eph-B2 on adhesion and migration of WM cells. Treating starved WM cells with recombinant ephrin-B2 (the ligand of Eph-B2) activated cell-adhesion signaling, including focal adhesion kinase (FAK), Src, p130, paxilin and cofilin, but had no effect on WM cell chemotaxis. Moreover, we found that ephrin-B2 was highly expressed on endothelial cells isolated from the BM of WM patients and on HUVEC cells. Inhibition of ephrin-B2 on the endothelial cells or Eph-B2 on the WM cells with blocking antibodies inhibited the adhesion of the WM cells to endothelial cells, as well as the cytoskeletal signaling in WM cells induced by co-culture with endothelial cells. Consequently, inhibition of the ephrin-B2/Eph-B2 interaction reversed the proliferative and cell cycle effect in WM cell which was induced by co-culture with endothelial cells. This was confirmed using BrdU proliferation assay, flow cytometry for cell cycle, and by immuno-blotting to confirm the effect on proliferative signaling pathways including PI3K kinase and cell cycle related proteins including Cyclin-D, Cyclin-E and p-Rb. Conclusion: This study examines the interaction of Eph-B2 receptor in WM and shows that ephrin- B2/Eph-B2 axis regulates adhesion, activation of downstream signaling of integrin-related molecules, survival and cell cycle progression through the interaction of tumor cells with endothelial cells. This provides the basis for further studies to explore Eph-B2 as a novel therapeutic target in WM. Disclosures: Ghobrial: Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Description: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment to regulate multiple cellular processes. Rapamycin and its analogs have not shown significant activity in multiple myeloma (MM), likely because of the lack of inhibition of TORC2. In the present study, we investigated the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. TORC1/2 knock-down led to significant inhibition of the proliferation of MM cells, even in the presence of BM stromal cells. We also tested INK128, a dual TORC1/2 inhibitor, as a new therapeutic agent against these MM cell lines. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin), even in the presence of cytokines or stromal cells. In vitro and in vivo studies showed that p-4EBP1 and p-Akt inhibition could be predictive markers of TORC2 inhibition in MM cell lines. Dual TORC1/2 inhibition showed better inhibition of adhesion to BM microenvironmental cells and inhibition of homing in vivo. These studies form the basis for further clinical testing of TORC1/2 inhibitors in MM.

Description: Abstract 3732 Poster Board III-668 Background Waldenstrom Macroglobulinema (WM) is an incurable B-cell disorder characterized by the presence of IgM monoclonal gammopathy and bone marrow infiltration of lymphoplasmacytic cells. Apart from promising advances in therapeutic strategy, response rates and treatment-free survival remain inconsistent across prescribed regimens. As we have previously shown, the PI3K/Akt pathway actively mediates tumor growth, survival, migration, and cell cycle within primary WM cells. An active target of this pathway includes the downstream mammalian target of rapamycin (mTOR). By inhibiting the mTOR pathway with RAD001 (Afinitor™, Novartis Pharmaceuticals), we hoped to constructively regulate aforementioned cellular function, and also understand from a preclinical perspective its specificity of action in our microenvironmental models, both alone and the presence of monoclonal antibody against CD20 and proteasome inhibitor. Methods WM cell lines (BCWM1) and IgM secreting cell lines MEC1 and RL were used. Bone marrow stromal cells (BMSC) were obtained from patients with WM. Peripheral blood mononuclear cells were harvested from healthy donors. Agents tested included RAD001, Rituximab and Bortezomib. Cytotoxicity, DNA synthesis, and cell cycle were measured using MTT assay, [3H]-thymidine uptake, and flow cytometry/PI staining, respectively. Antibody-dependent cellular cytoxicity (ADCC), transwell, and Matrigel assays were preformed to measure cell lysis, SDF-1 chemotaxis-induced migration, and angiogenesis, respectively. Adhesion to fibronectin has been evaluated with WM cells and BMSC in the presence of RAD001, with and without Bortezomib. Results RAD001 induced cytotoxicity and inhibition of DNA synthesis with an IC50 of 1-10 nM in BCWM1 cells at 48 hours. Similar effects were seen in IgM cell lines with an effective dose of 0.1-1 nM. In contrast, at IC50 treatment level donor cells displayed no significant cytotoxicity (〉85% survival). Cell cycle analysis showed corresponding G1 arrest, and angiogenesis was also inhibited. RAD001, Bortezomib, and Rituxan combinations showed synergistic cytotoxicity, which was attenuated when WM cells were co-cultured with BMSC. Migration of BCWM1 and adhesion of BCWM1 to BMSC was reduced under presence of RAD001. Conclusions These functional assays therefore show that RAD001 has significant and synergistic antitumor activity in WM which will inform the design of future clinical trials. Disclosures: Ghobrial: Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Description: Abstract 625 Background. Bone marrow (BM)-derived mesenchymal stem cells (MSCs) support multiple myeloma (MM) cell growth, but little is know about the putative mechanisms that may regulate the interaction between clonal MM plasma cells and the surrounding BM milieu. It is known that cell-to-cell communication is partially mediated by exosomes. We therefore characterized the role BM-MSCs-derived exosomes as key regulators of MM pathogenesis in vivo and in vitro. Methods. MSCs were collected from BM of either healthy subjects or relapsed/refractory MM patients. MM cell lines (MM.1S; RPMI.8226) and normal BM stromal cell line (HS-5) were used. Purity of BM-MSCs was evaluated by flow cytometry (CD34−,14−, 45−, 19−, 138−; CD73+, 90+, 105+, 106+). Exosomes were collected from conditioned medium of either normal and MM BM-MSCs, or HS-5 cells; and studied using electron microscopy, immunogold labeling, and western blot for the detection of CD63 and CD81. Transfer of PKH67-fluorescently labeled exosomes to MM cells was evaluated by confocal microscopy and fluorescence plate reader. Transfer of murine-derived miRNA-containing exosomes into human MM cell lines was evaluated by qRT-PCR (exosomes were collected from BM-MSCs of C57BL/6 miRNA-15a/16-1−/− or C57BL/6 mice). miRNA expression profiling was obtained from normal (n=4) and MM (n=9) BM-MSCs-derived exosomes (TaqMan human miRNA profiling). Normal and MM BM-MSCs-derived exosomes were loaded into tissue-engineered bones (TEB) with MM.1S-GFP+/Luc+ cells: MM cell homing and MM tumor growth has been tested in vivo by using in vivo confocal microscopy and bioluminescence (BLI) imaging, respectively. Normal and MM BM-MSCs, as well as HS-5 cells, were transfected with either anti- or pre-miRNA-15a or scramble probe; and evaluated for their ability to modulate MM cell proliferation and adhesion in vitro. Results. Primary normal and MM BM-MSCs release CD63+/CD81+ exosomes, as confirmed by electron microscopy, immunogold labeling, and western blot. BM-MSCs exosomes are transferred into MM cells, as shown by confocal microscopy. This transfer was further confirmed in human MM cell lines incubated with murine (C57BL/6 miRNA-15a/16-1−/− and wild type) BM-MSCs-derived exosomes: qRT-PCR showed presence of murine miRNAs in human MM cell lines. The impact of normal and MM BM-MSCs-derived exosomes on MM cell behavior in vivo was next evaluated. MM cells co-cultured with exosomes derived from MM BM-MSCs induced rapid tumor growth at the site of the TEB scaffold, as well as rapid dissemination in the BM niches. In contrast, MM cells co-cultured with exosomes derived from normal BM-MSCs led to minimal tumor growth and minimal dissemination at distant BM niches. These results indicate that MM BM-MSCs-derived exosomes contribute to tumor growth and dissemination of MM. To further explore the mechanisms by which exosomes induce tumor growth, we performed miRNA expression profiling on exosomes isolated from both normal and MM BM-MSCs: supervised hierarchical clustering analysis showed increased expression of 24 miRNAs and reduced expression of 3 miRNAs in MM BM-MSCs-derived exosomes versus normal (1.5 fold change; P

Description: Abstract 440 Background. Multiple myeloma (MM) patients present with multiple lytic lesions at diagnosis, indicating the presence of continuous dissemination of MM cells from the primary site of tumor development to multiple distant bone marrow (BM) niches. We hypothesized that stromal-derived factor-1 (SDF-1) may represent a target for preventing transition from MGUS (micrometastatic stage) to active-MM (macrometastatic stage); thus resulting in inhibition of MM progression. We therefore evaluated SDF-1 expression in the BM of patients with MGUS, MM, compared to healthy individuals; and tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1 in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vitro and in vivo . Methods. SDF-1 levels were evaluated by immunohistochemistry on BM specimens obtained from patients with MGUS, active-MM, or healthy individuals; and confirmed by ELISA, using conditioned-medium of BM-mesenchymal stromal cells obtained from MGUS, active-MM and healthy individuals. BM metastatic lesions from primary epithelial tumors were also considered. Co-localization of MM tumor cells (MM.1S-GFP+) with SDF-1 was tested in vivo by in vivo confocal microscopy, using both AlexaFluor633-conjugated-anti-SDF-1 monoclonal antibody and AlexaFluor647-conjugated-NOX-A12 oligonucleotide. Effect of NOX-A12 on modulating MM cell dissemination was tested in vivo, by using in vivo confocal microscopy. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) were assessed by using in vivo confocal microscopy and in vivo bioluminescence, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12+bortezomib. Detection of mobilized MM-GFP+ cells ex vivo was performed by flow cytometry. Effects of drug combination on dissemination of MM cells to distant BM niches was evaluated ex vivo by immunofluorescence on femurs obtained from each cohort of mice. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–100nM) treated primary MM BM stromal cells (BMSCs) in presence or absence of bortezomib (2.5–5nM) were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software. NOX-A12-dependent-modulation of signaling was evaluated by western blot on MM cells exposed or not to primary BM-MSCs. Results. Patients with active-MM present with higher BM SDF-1 expression vs. MGUS patients and healthy individuals. Similarly, BM presenting with metastasis from epithelial primary malignancies had higher SDF-1 levels compared to healthy subjects, thus suggesting the importance of SDF-1 in favoring tumor cell metastasis to BM niches. SDF-1 co-localized at BM level with MM tumor cells in vivo. In vitro, NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BMSCs supported by inhibition of BM-MSC-mediated phosphorylation of ERK1/2 and cofilin. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the peripheral blood of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). This was supported by inhibited homing of MM cells to the BM of those mice pre-treated with NOX-A12. We next showed that NOX-A12-dependent de-adhesion of MM cells from BMSCs lead to enhanced MM cell sensitivity to bortezomib, as shown in vitro, where a synergistic effect between NOX-A12 and bortezomib was observed (C.I.: .57-.76). These findings were validated in vivo: tumor burden was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P