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Supplementary Figures S1-S3.

Purpose: Primary Waldenstrom's Macroglobulinemia (WM) cells present with a significantly higher level of the immunoproteasome compared with the constitutive proteasome. It has been demonstrated that selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive-(c20S) and immuno-(i20S) proteasome represents a valid strategy to induce antineoplastic effect in hematologic tumors. We therefore evaluated carfilzomib, a potent selective, irreversible inhibitor of the CT-L activity of the i20S and c20S in WM cells.Experimental Design: We tested the effect of carfilzomib on survival and proliferation of primary WM cells, as well as of other IgM-secreting lymphoma cell lines. Carfilzomib-dependent mechanisms of induced apoptosis in WM cells, and its effect on WM cells in the context of bone marrow (BM) microenvironment have been also evaluated. Moreover, the combinatory effect of carfilzomib and bortezomib has been investigated. In vivo studies have been performed.Results: We demonstrated that carfilzomib targeted the CT-L activity of both i20S and c20S, which led to the induction of toxicity in primary WM cells, as well as in other IgM-secreting lymphoma cells. Importantly, carfilzomib targeted WM cells even in the context of BM milieu. In addition, carfilzomib induced apoptosis through c-jun-N-terminal-kinase activation, caspase cleavage, and initiation of unfolded protein response. Importantly, the combination of carfilzomib and bortezomib synergistically inhibited CT-L activity, as well as caspase-, PARP-cleavage and GRP94 expression. Antitumor activity of carfilzomib has been validated in vivo.Conclusions: These findings suggest that targeting i20S and c20S CT-L activity by carfilzomib represents a valid antitumor strategy in WM and other IgM-secreting lymphomas. Clin Cancer Res; 17(7); 1753–64. ©2011 AACR.

Boesenbergia pandurata (Roxb.) Schltr. is a perennial medicinal herb belonging to the Zingiberaceae family. It is cultivated in some tropical countries in Southeast Asia including Vietnam, Thailand, Myanmar, Indonesia and Malaysia. In Vietnam, it is known as "Ngai bun", and the fresh rhizomes are mainly used as a spice. This plant contains prenylated chalcones and other flavonoids as the major bioactive constituents. From the CHCl3 extract of Boesenbergia pandurata, we had isolated six flavanones. Their structures, pinocembrin (1), pinostrobin (2), alpinetin (3), di-Omethylpinocembrin (4), naringenin (5) and 5-Omethylnaringenin (6), were determined based on extensive spectroscopic analysis and in comparison with previous works.

The methylthioadenosine phosphorylase (MTAP) gene is located adjacent to the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor-suppressor gene and is co-deleted with CDKN2A in approximately 15% of all cancers. This co-deletion leads to aggressive tumors with poor prognosis that lack effective, molecularly targeted therapies. The metabolic enzyme methionine adenosyltransferase 2α (MAT2A) was identified as a synthetic lethal target in MTAP-deleted cancers. We report the characterization of potent MAT2A inhibitors that substantially reduce levels of S-adenosylmethionine (SAM) and demonstrate antiproliferative activity in MTAP-deleted cancer cells and tumors. Using RNA sequencing and proteomics, we demonstrate that MAT2A inhibition is mechanistically linked to reduced protein arginine methyltransferase 5 (PRMT5) activity and splicing perturbations. We further show that DNA damage and mitotic defects ensue upon MAT2A inhibition in HCT116 MTAP-/- cells, providing a rationale for combining the MAT2A clinical candidate AG-270 with antimitotic taxanes.

SummaryHomozygous deletions of p16/CDKN2A are prevalent in cancer, and these mutations commonly involve co-deletion of adjacent genes, including methylthioadenosine phosphorylase (MTAP). Here, we used shRNA screening and identified the metabolic enzyme, methionine adenosyltransferase II alpha (MAT2A), and the arginine methyltransferase, PRMT5, as vulnerable enzymes in cells with MTAP deletion. Metabolomic and biochemical studies revealed a mechanistic basis for this synthetic lethality. The MTAP substrate methylthioadenosine (MTA) accumulates upon MTAP loss. Biochemical profiling of a methyltransferase enzyme panel revealed that MTA is a potent and selective inhibitor of PRMT5. MTAP-deleted cells have reduced PRMT5 methylation activity and increased sensitivity to PRMT5 depletion. MAT2A produces the PRMT5 substrate S-adenosylmethionine (SAM), and MAT2A depletion reduces growth and PRMT5 methylation activity selectively in MTAP-deleted cells. Furthermore, this vulnerability extends to PRMT5 co-complex proteins such as RIOK1. Thus, the unique biochemical features of PRMT5 create an axis of targets vulnerable in CDKN2A/MTAP-deleted cancers.

Nature 547, 109–113 (2017); doi:10.1038/nature22964 In this Letter, there are errors in Extended Data Figs 5, 8 and 9, and the affiliation of an author. The affiliations for author Violeta Serra should include number 2 (CIBERONC, Instituto de Salud Carlos III, C/ Monforte de Lemos 3-5, Pabellon 11, Planta 0, 28029 Madrid, Spain).

License Plate Recognition is a practical use of computer vision based application. With the increase in demand of automation transportation systems, this application plays a very big role in the system development. Also, the use of vehicles has been increasing because of population growth and human needs in recent years makes the application is more challenging. Moreover, license plates are available in diverse colors and style and that the presence of noise, blurring in the image, uneven illumination, and occlusion makes the task even more difficult for conventional recognition methods. We propose an approach of using a Convolutional Neural Networks (CNN) classifier for the recognition. Pre-processing techniques are firstly applied on input images, such as filtering, thresholding, and then segmentation. Then, we train a CNN classifier for character recognition. Although the performance of a CNN is very impressive, it costs much time to complete the character recognition step. In this study, a modified CNN is proposed to help the system run in real-time. Experimental results have done and analyzed with other methods.

Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation,. Here we show that mTORC1 regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. Through the use of integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identified alterations in tumours impacting on the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation was validated in murine and human cancer specimens. AMD1 was upregulated in prostate cancer specimens with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibited a predominant decrease in AMD1 immunoreactivity that was associated to a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.

Tetrad test is theoretically more powerful than triangle test. However, in the presence of sensory fatigue-caused foods, the advantage of the tetrad test is lost. In case of alcohol beverage containing 50%abv, triangle test is recommended as a standard protocol because the sensory fatigue caused by ethanol is great enough that leads tetrad test be less powerful than triangle test. In this study, the association between alcohol concentration and power of tetrad as well as triangle was established in cases of alcoholic beverages lower than 50%abv. Based on this result, the alcohol concentration at which the tetrad test is more powerful than triangle test was determined. The power comparisons between triangle test and tetrad test were extended to some alcohol beverages containing 40 and 30%abv. 240 panelists were divided into 8 panels. In each session, both triangle test and tetrad test were performed by a same panel (N = 30) on the samples made from the same alcohol beverage basic with and without citric acid. There were four sessions for each product that corresponded with four increasingly levels of added citric acid (2.192, 3.1, 4.384, 6.2g/L). By applying logistic regression, the power comparison between two tests was based on the correct response proportions (Pc) which were predicted from two variables: acid concentration and protocol. Multivariate logistic regression analysis indicated that there was 84% chance and 38% chance for the tetrad’s Pc to be higher than triangle’s Pc in case of beverage containing 30 and 40%abv, respectively. The results shown that increase alcohol concentration will reduce the power of both triangle and tetrad tests. It was noteworthy that tetrad test was affected more strongly by alcohol than triangle test. These findings suggest that the triangle test can be used as a suitable testing methodology for alcoholic beverages which alcohol concentration is not lower than 40%abv. In case of beverage containing 30%abv, triangle test should be replaced by tetrad test due to the increased reliability of information.

The spread of multiple myeloma (MM) involves (re)circulation into the peripheral blood and (re)entrance or homing of MM cells into new sites of the BM. Hypoxia in solid tumors was shown to promote metastasis through activation of proteins involved in the epithelial-mesenchymal transition (EMT) process. We hypothesized that MM-associated hypoxic conditions activate EMT-related proteins and promote metastasis of MM cells. In the present study, we have shown that hypoxia activates EMT-related machinery in MM cells, decreases the expression of E-cadherin, and, consequently, decreases the adhesion of MM cells to the BM and enhances egress of MM cells to the circulation. In parallel, hypoxia increased the expression of CXCR4, consequently increasing the migration and homing of circulating MM cells to new BM niches. Further studies to manipulate hypoxia to regulate tumor dissemination as a therapeutic strategy are warranted.

Purpose: There is no standard of therapy for the treatment of Waldenström macroglobulinemia (WM), therefore there is a need for the development of new agents. Fibroblast growth factor receptor 3 (FGFR3) was shown to play a major role in several types in cancer. Dovitinib, an inhibitor of FGFR3, was effective in hematologic malignancies. In this study, we tested FGFR3 as a therapeutic target in WM and tested the effect of dovitinib on cell proliferation and apoptosis of WM cells in the context of BM microenvironment. Methods: The expression of FGFR3 in WM cells was tested using immunofluorescence and flow cytometry. Cell signaling in response to stimulation with FGF3 and stromal cells, and its inhibition by dovitinib was performed using immunoblotting. Cell survival and cell proliferation were assessed by MTT and BrdU assays. Apoptosis was measured by detection of APO-2.7 and cleavage of caspase-3 using flow cytometry. Cell cycle was performed by PI staining of cells and flow cytometry. The combinatory effect of dovitinib with other drugs was analyzed using Calcusyn software. The effect of dovitinib was tested in vivo. Results: FGFR3 was overexpressed in WM cells and its activation induced cell proliferation. Inhibition of FGFR3 with dovitinib decreased cell survival, increased apoptosis, and induced cell cycle arrest. Inhibition of FGFR3 by dovitinib reduced the interaction of WM to bone marrow components, and reversed its proliferative effect. Dovitinib had an additive effect with other drugs. Moreover, dovitinib reduced WM tumor progression in vivo. Conclusion: We report that FGFR3 is a novel therapeutic target in WM, and suggest dovitinib for future clinical trial the treatment of patients with WM. Clin Cancer Res; 17(13); 4389–99. ©2011 AACR.

Waldenstrom macroglobulinemia (WM) is a low-grade B-cell lymphoproliferative disorder characterized primarily by specific homing and growth of tumor cells within the bone marrow niches. The progressive growth of tumor cells throughout the bone marrow indicates that the tumor cells are capable of homing and adhering to specific niches that allow growth, survival and drug resistance. In this review we highlight the interaction of the tumor cells in WM and the bone marrow microenvironment including bone marrow stromal cells, endothelial cells and mast cells. Migration, adhesion and downstream activation of signaling pathways leads to cell trafficking and cell dissemination in WM. Future therapeutic agents need to target not only the tumor clone, but also its close interaction with the bone marrow microenvironment.

Waldenström macroglobulinemia (WM) cells present with increased expression of microRNA-206 (miRNA-206) and reduced expression of miRNA-9*. Predicted miRNA-206– and -9*–targeted genes include histone deacetylases (HDACs) and histone acetyl transferases (HATs), indicating that these miRNAs may play a role in regulating histone acetylation. We were able to demonstrate that primary WM cells are characterized by unbalanced expression of HDACs and HATs, responsible for decreased acetylated histone-H3 and -H4, and increased HDAC activity. We next examined whether miRNA-206 and -9* modulate the aberrant expression of HDAC and HATs in WM cells leading to increased transcriptional activity. We found that restoring miRNA-9* levels induced toxicity in WM cells, supported by down-modulation of HDAC4 and HDAC5 and up-regulation of acetyl-histone-H3 and -H4. These, together with inhibited HDAC activity, led to induction of apoptosis and autophagy in WM cells. To further confirm that miRNA-9*–dependent modulation of histone acetylation is responsible for induction of WM cytotoxicity, a novel class of HDAC inhibitor (LBH589) was used; we confirmed that inhibition of HDAC activity leads to toxicity in this disease. These findings confirm that histone-modifying genes and HDAC activity are deregulated in WM cells, partially driven by the aberrant expression of miRNA-206 and -9* in the tumor clone.

Proteasome inhibition represents a valid antitumor approach and its use has been validated in Waldenström macroglobulinemia (WM), where bortezomib has been successfully tested in clinical trials. Nevertheless, a significant fraction of patients relapses, and many present toxicity due to its off-target effects. Selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive proteasome 20S (c20S) and immunoproteasome 20S (i20S) represents a sufficient and successful strategy to induce antineoplastic effect in hematologic tumors. We therefore studied ONX0912, a novel selective, irreversible inhibitor of the CT-L activity of i20S and c20S. Primary WM cells express higher level of i20S compared with c20S, and that ONX0912 inhibited the CT-L activity of both i20S and c20S, leading to induction of toxicity in primary WM cells, as well as of apoptosis through c-Jun N-terminal kinase activation, nuclear factor κB (NF-κB) inhibition, caspase cleavage, and initiation of the unfolded protein response. Importantly, ONX0912 exerted toxicity in WM cells, by reducing bone marrow (BM)–derived interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1) secretion, thus inhibiting BM-induced p-Akt and phosphorylated extracellular signal-related kinase (p-ERK) activation in WM cells. These findings suggest that targeting i20S and c20S CT-L activity by ONX0912 represents a valid antitumor therapy in WM.

Alcohols are considered environmentally benign alkylating agents since the only by-product generated in their reactions is water. Herein, we report a microwave-assisted Friedel―Crafts alkylation and electrophilic annulation of indoles using alcohols as alkylating agents. Alkylation of indoles using tert-butyl alcohol gives 3-substituted tert-butylindoles. A domino electrophilic annulation/ aromatization of indoles using hexane-2,5-diol results in the formation of substituted carbazoles. The reaction is catalyzed by a strong, solid-acid catalyst montmorillonite K-10. The products were obtained in good yields and high selectivities.

The widespread involvement of the bone marrow with tumor cells indicates that there is continuous cell trafficking of WM cells in and out of the bone marrow leading to cell dissemination in the bone marrow and in the lymph nodes in many patients with WM. The interaction of the WM cells with the bone marrow is critical for the regulation of cell proliferation, cell cycle, drug resistance as well as cell dissemination and trafficking. Advances in understanding the interaction of the tumor clone with the BM microenvironment have led to the development of therapeutic agents that not only target the tumor clone but also regulate the bone marrow microenvironment. Here, we review the role of the cellular and liquid bone marrow compartments in the regulation of cell proliferation and dissemination in WM.

Epigenetics represent heritable changes in gene expression that are not due to any alteration in the DNA sequence. One of the best-known epigenetic markers is histone acetylation, which has been shown to be deregulated in neoplastic diseases, including B-cell malignancies, such as Waldenström's Macroglobulinemia (WM), a low-grade B-cell lymphoma characterized by the presence of lymphoplasmacytic cells in the bone marrow and a serum monoclonal immunoglobulin M in the circulation. It has been recently demonstrated that microRNAs may be responsible for modulating histone acetylation in WM cells, thus providing the preclinical evidences for using microRNA-based therapeutic strategies in this disease.

Phospholipase D enzymes cleave lipid substrates to produce phosphatidic acid, an important precursor for many essential cellular molecules. Phospholipase D is a target to modulate cancer-cell invasiveness. This study reports synthesis of a new class of phospholipase D inhibitors based on 1,3-disubstituted-4-amino-pyrazolopyrimidine core structure. These molecules were synthesized and used to perform initial screening for the inhibition of purified bacterial phospholipase D, which is highly homologous to the human PLD1 . Initially tested with the bacterial phospholipase D enzyme, then confirmed with the recombinant human PLD1 and PLD2 enzymes, the molecules presented here exhibited inhibition of phospholipase D activity (IC50 ) in the low-nanomolar to low-micromolar range with both monomeric substrate diC4 PC and phospholipid vesicles and micelles. The data strongly indicate that these inhibitory molecules directly block enzyme/vesicle substrate binding. Preliminary activity studies using recombinant human phospholipase Ds in in vivo cell assays measuring both transphosphatidylation and head-group cleavage indicate inhibition in the mid- to low-nanomolar range for these potent inhibitory novel molecules in a physiological environment.

Glucocorticoid-induced TNF receptor (GITR) plays a crucial role in modulating immune response and inflammation, however the role of GITR in human cancers is poorly understood. In this study, we demonstrated that GITR is inactivated during tumor progression in Multiple Myeloma (MM) through promoter CpG island methylation, mediating gene silencing in primary MM plasma cells and MM cell lines. Restoration of GITR expression in GITR deficient MM cells led to inhibition of MM proliferation in vitro and in vivo and induction of apoptosis. These findings were supported by the presence of induction of p21 and PUMA, two direct downstream targets of p53, together with modulation of NF-κB in GITR-overexpressing MM cells. Moreover, the unbalanced expression of GITR in clonal plasma cells correlated with MM disease progression, poor prognosis and survival. These findings provide novel insights into the pivotal role of GITR in MM pathogenesis and disease progression.

miR-155 acts as an oncogenic miR in B-cell lymphoproliferative disorders, including Waldenstrom macroglobulinemia (WM) and chronic lymphocytic leukemia, and is therefore a potential target for therapeutic intervention. However, efficient targeting of miRs in tumor cells in vivo remains a significant challenge for the development of miR-155–based therapeutics for the treatment of B-cell malignancies. In the present study, we show that an 8-mer locked nucleic acid anti–miR-155 oligonucleotide targeting the seed region of miR-155 inhibits WM and chronic lymphocytic leukemia cell proliferation in vitro. Moreover, anti–miR-155 delivered systemically showed uptake in the BM CD19+ cells of WM-engrafted mice, resulting in the up-regulation of several miR-155 target mRNAs in these cells, and decreased tumor growth significantly in vivo. We also found miR-155 levels to be elevated in stromal cells from WM patients compared with control samples. Interestingly, stromal cells from miR-155–knockout mice led to significant inhibition of WM tumor growth, indicating that miR-155 may also contribute to WM proliferation through BM microenvironmental cells. The results of the present study highlight the therapeutic potential of anti–miR-155–mediated inhibition of miR-155 in the treatment of WM.

The spread of multiple myeloma (MM) involves (re)circulation into the peripheral blood and (re)entrance or homing of MM cells into new sites of the BM. Hypoxia in solid tumors was shown to promote metastasis through activation of proteins involved in the endothelial to mesenchymal transition (EMT) process. In this study, we hypothesized that MM associated hypoxic conditions activates EMT related proteins and promotes metastasis of MM cells. Hypoxia activated EMT-related machinery in MM cells, decreased expression of E-cadherin and consequently decreased adhesion of MM cells to the BM and enhanced egress of MM cells to the circulation. In parallel, hypoxia increased the expression of CXCR4, and consequently increased the migration and homing of circulating MM cells to new BM niches. Further studies to manipulate hypoxia in order to regulate tumor dissemination as a therapeutic strategy are warranted.

Interactions between multiple myeloma (MM) cells and the BM microenvironment play a critical role in the pathogenesis of MM and in the development of drug resistance by MM cells. Selectins are involved in extravasation and homing of leukocytes to target organs. In the present study, we focused on adhesion dynamics that involve P-selectin glycoprotein ligand-1 (PSGL-1) on MM cells and its interaction with selectins in the BM microenvironment. We show that PSGL-1 is highly expressed on MM cells and regulates the adhesion and homing of MM cells to cells in the BM microenvironment in vitro and in vivo. This interaction involves both endothelial cells and BM stromal cells. Using loss-of-function studies and the small-molecule pan-selectin inhibitor GMI-1070, we show that PSGL-1 regulates the activation of integrins and downstream signaling. We also document that this interaction regulates MM-cell proliferation in coculture with BM microenvironmental cells and the development of drug resistance. Furthermore, inhibiting this interaction with GMI-1070 enhances the sensitization of MM cells to bortezomib in vitro and in vivo. These data highlight the critical contribution of PSGL-1 to the regulation of growth, dissemination, and drug resistance in MM in the context of the BM microenvironment.

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.

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.

Purpose: The ephrin receptors (Eph) are found in a wide range of cancers and correlate with metastasis. In this study, we characterized the role of Eph-B2 receptor in the interaction of Waldenstrom's macroglobulinemia (WM) cells with the bone marrow microenvironment. Experimental Design: We screened the activity of different receptor tyrosine kinases in WM patients and found that Eph-B2 was overexpressed compared with control. Also, we tested the expression of ephrin-B2 ligand on endothelial cells and bone marrow stromal cells (BMSC) isolated from WM patients. We then tested the role of Eph-B2/Ephrin-B2 interaction in the adhesion of WM cells to endothelial cells and BMSCs; the cell signaling induced by the coculture in both the WM cells and the endothelial cells; WM cell proliferation, apoptosis, and cell cycle in vitro and tumor progression in vivo; and in angiogenesis. Results: Eph-B2 receptor was found to be activated in WM patients compared with control, with a 5-fold increase in CD19+ WM cells, and activated cell adhesion signaling, including focal adhesion kinase, Src, P130, paxillin, and cofilin, but decreased WM cell chemotaxis. Ephrin-B2 ligand was highly expressed on endothelial cells and BMSCs isolated from WM patients and on human umbilical vein endothelial cells and induced signaling in the endothelial cells promoting adhesion and angiogenesis. Blocking of ephrin-B2 or Eph-B2 inhibited adhesion, cytoskeletal signaling, proliferation, and cell cycle in WM cells, which was induced by coculture with endothelial cells and decreased WM tumor progression in vivo. Conclusion: Ephrin-B2/Eph-B2 axis regulates adhesion, proliferation, cell cycle, and tumor progression in vivo through the interaction of WM with the cells in the bone marrow microenvironment. Clin Cancer Res; 18(1); 91–104. ©2011 AACR.

Waldenstrom macroglobulinemia (WM) is a low-grade B-cell lymphoma characterized by widespread involvement of the bone marrow with lymphoplasmacytic cells. In approximately 20% of patients, the malignant clone also involves the lymph nodes and induces hepatosplenomegaly. The mechanisms by which the tumor cells home to the bone marrow and preferentially reside in the marrow niches are not fully elucidated. In this review, we examine the role of the bone marrow microenvironment in the regulation of cell growth, survival and cell dissemination in WM. We also summarize specific regulators of niche-dependent tumor proliferation in WM. These include chemokines, adhesion molecules, Src/PI3K/Akt/mTOR signaling, NF-kB activation, and micro-RNA regulation in WM. Targeting these pathways in clinical trials could lead to significant responses in this rare disease.

We have previously shown clinical activity of a mammalian target of rapamycin (mTOR) complex 1 inhibitor in Waldenstrom macroglobulinemia (WM). However, 50% of patients did not respond to therapy. We therefore examined mechanisms of activation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR in WM, and mechanisms of overcoming resistance to therapy. We first demonstrated that primary WM cells show constitutive activation of the PI3K/Akt pathway, supported by decreased expression of phosphate and tensin homolog tumor suppressor gene (PTEN) at the gene and protein levels, together with constitutive activation of Akt and mTOR. We illustrated that dual targeting of the PI3K/mTOR pathway by the novel inhibitor NVP-BEZ235 showed higher cytotoxicity on WM cells compared with inhibition of the PI3K or mTOR pathways alone. In addition, NVP-BEZ235 inhibited both rictor and raptor, thus abrogating the rictor-induced Akt phosphorylation. NVP-BEZ235 also induced significant cytotoxicity in WM cells in a caspase-dependent and -independent manner, through targeting the Forkhead box transcription factors. In addition, NVP-BEZ235 targeted WM cells in the context of bone marrow microenvironment, leading to significant inhibition of migration, adhesion in vitro, and homing in vivo. These studies therefore show that dual targeting of the PI3K/mTOR pathway is a better modality of targeted therapy for tumors that harbor activation of the PI3K/mTOR signaling cascade, such as WM.

Within the past few years, major advances in the preclinical and clinical testing of novel therapeutic agents have occurred in Waldenstrom's macroglobulinemia (WM). These include agents that target the PI3K/Akt/mTOR pathway, PKC pathways, NF-kB signaling pathway, as well as tyrosine kinases and histone deacetylase inhibitors. In this review, we summarize the current understanding of the clinical development of these agents in WM.

B570 Centrosomal Clustering: A Novel Therapeutic Target for Multiple Myeloma MS Raab, I Breitkreutz, B Rebacz, PJ Hayden, TO Larsen, MH Clausen, JH Fruehauf, H Goldschmidt, KC Anderson, A Kraemer Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; CCU Molecular Hematology/ Oncology, German Cancer Research Center, Heidelberg, Germany; BioCentrum, Technical University of Denmark, Kopenhagen, Denmark; Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; National Center of Tumor Diseases, Dept. of Medicine, University of Heidelberg, Germany

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 Conclusion. SDF-1 represents a valid target for inhibiting MM cell dissemination to distant BM niches, thus providing the evidence for using the SDF-1 inhibiting Spiegelmer NOX-A12 to target MM cells at the stage of micrometastasis (MGUS), thus preventing development of symptomatic macrometastatic MM. Disclosures: Zboralski: NOXXON Pharma AG, Berlin, Germany: Employment. Kruschinski:NOXXON Pharma AG, Berlin, Germany: Employment. Ghobrial:Novartis: Advisory Board Other; Onyx: Advisory Board, Advisory Board Other; Millennium: Advisory Board, Advisory Board Other; Bristol Myers Squibb: Advisory Board, Advisory Board Other.

Abstract 3669 Background MicroRNAs (miRNAs) are short, non-coding RNA molecules that average 18–24 nucleotides long and act as important post-transcriptional regulators. It has been demonstrated that miRNAs stably circulate in the blood and can be detected in plasma and serum. Numerous disease states, including both solid tumor and hematologic malignancies, have been shown to release miRNAs into the circulation. Furthermore, these tumor cells possess their own characteristic expression patterns of miRNAs. This presents an opportunity for specific miRNAs to serve as biomarkers for different diseases. Previous studies have shown microRNA-155 (miR-155) to be elevated in low grade lymphomas such as Chronic Lymphocytic Leukemia (CLL) and Waldenstrom Macroglobulinemia (WM). Currently, an established biomarker for diagnostic or prognostic applications does not exist for these diseases. In our study, we investigate miR-155 as a potential biomarker for CLL and WM in plasma. Methods Both plasma and serum samples were collected from CLL patients, WM patients, and healthy individuals. In order to quantify miR-155 levels, 100 μl of each sample went through a round of RNA isolation, reverse transcription (RT) reaction, and real-time quantitative PCR (qPCR) by using miRNeasy mini kit (Qiagen), Taqman microRNA reverse transcription kit (Applied Biosystem) and RT2 Fast SYBR® Green/ROX qPCR Master Mixes (SABiosciences). Based on the RT-qPCR results, samples were classified as having either detectable miR-155 (Ct value less than 40) or undetectable miR-155 (Ct value more than 40). The ratio of detectable to undetectable samples was used to compare groups of samples: i) CLL plasma, ii) CLL serum, iii) WM plasma, iv) WM serum v) Normal plasma, and vi) Normal serum. To adjust for variations in efficiency of RNA extraction among samples, C. elegans synthetic microRNA-39 was added specifically after denaturation of the sample and used as a spiked-in control. By using the CD63 antigen as a marker, exosomes in the plasma and serum were also detected through flow cytometry. Results A total of 20 CLL plasma samples showed a 100% positive ratio of detectable miR-155 while 20 CLL serum samples had only a 50% positive ratio. In addition, a total of 96 WM plasma samples had a positive ratio of 74% while 30 WM serum samples showed a much lower positive ratio of 3%. Of these 96 samples, 13 were obtained from upfront WM patients while 83 were from relapsed/refractory WM patients. The 10 plasma and 10 serum samples from healthy individuals were all negative for miR-155. Lastly, preliminary data indicated that a higher level of exosomes may exist in plasma compared to serum. Summary This finding suggests that miR-155 in peripheral blood plasma could potentially be used as a clinical biomarker for WM and/or CLL. The considerable difference in detectable miR-155 between patient plasma and serum was unexpected. Reasons for this disparity are still under investigation. Our preliminary data indicated that this may be due to a difference in exosomes, which are known to carry miRNAs, between plasma and serum. Ultimately, further investigation is warranted within a larger cohort to confirm the status of miR-155 as a reliable plasma biomarker for these low grade B cell malignancies. Disclosures: Roccaro: Roche. Ghobrial:Novartis: Consultancy, 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.

Abstract 3934 INTRODUCTION: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis. The BM microenvironment in MM is characterized by an increased micro-vessel density and increased secretion of angiogenic factors. CXCR7 is a G-protein coupled receptor shown to play a major role in the adhesion, migration and angiogenesis of endothelial cells (ECs). Our interest is in the role of CXCR7 in cell trafficking of ECs and EPCs in MM. Thus we characterized ECs and EPCs from MM patients and MM animal models and examined the contribution of CXCR7 to the cell trafficking using in vitro and in vivo assays and using CXCR7-selective compound. METHODS AND RESULTS: We used flow cytometry to detect the frequency of ECs and EPCs in the BM and peripheral blood (PB) of 5 MM patients and 5 normal subjects. ECs were detected as VEGFR+ CD133- cells, while EPCs were detected as VEGFR+ CD133+ cells. MM patients had significantly higher numbers of ECs and EPCs compared to healthy donors in both the BM and the PB. These results were confirmed in a mouse model of MM in which MM cells or vehicle were injected to SCID mice and the frequency of ECs and EPCs in the BM and the PB was determined 4 weeks after injection. We found that in mice with MM significantly higher numbers of ECs and EPCs could be detected in both the BM and the PB than in control mice. CXCR7 was expressed on both ECs and EPCs isolated from MM patients, healthy donors, and control mice. The expression of CXCR7 on EPCs was higher than the expression on ECs. The expression of CXCR7 on ECs and EPCs isolated from the BM was higher than the expression on ECs and EPCs isolated from the PB, respectively. Therefore, to test the role of CXCR7 in cell-trafficking of ECs and EPCs, we injected 10mg/kg of CXCR7 inhibitor POL6926, a potent and selective CXCR7 antagonist based on the Protein Epitope Mimetics (PEM) Technology (Polyphor, Switzerland), to BALB/c mice and tested the frequency of ECs and EPC in the PB and BM of the mice at 0, 2, 4 and 24 hours after the injection. We found a 3-fold increase in ECs and 6-fold increase in EPCs in the PB; 2 hrs post the injection of the CXCR7 antagonist. The levels of EPCs in the PB returned to baseline at 4 and 24 hrs, while the level of ECs was maintained at 4hrs and went back to baseline at 24 hrs. No significant differences were found in the frequency of ECs and EPCs in the BM after the injection of the CXCR7 antagonist. To investigate the function of CXCR7 in ECs in vitro we used human umbilical vein endothelial cells (HUVECs) as a model for ECs. CXCR7 was highly expressed on HUVECs. We could demonstrate that in vitro tube formation was promoted by either co-culture of MM cells or by conditioned medium from MM cell cultures. Furthermore, migration of HUVEC cells was facilitated by conditioned medium from MM cell cultures. These data suggest that MM cells may secrete factors promoting migration of endothelial cells and pro-angiogenic factors promoting angiogenesis. In addition, we could show that in vitro tube formation is inhibited by POL6926 suggesting that CXCR7 expression on HUVECs is required for tube formation. At the test concentrations POL6926 was not cytotoxic to HUVECs since cell proliferation was unaffected. CONCLUSION: We have shown that the level of ECs and EPCs was elevated in the PB and BM of MM patients compared to normal subjects, a finding which was confirmed in a MM mouse model in which CXCR7 was highly expressed on these cells. Injection of PEM CXCR7 antagonist increased the numbers of ECs and EPCs in the PB. These results suggest that CXCR7 may play a role in the cell-trafficking and recruitment of ECs and EPCs in MM. To investigate this hypothesis, using in vitro tube formation and migration assays, we have shown that MM cells secrete factors that promote migration and angiogenesis of HUVECs and the PEM CXCR7 antagonist inhibits these processes. In subsequent studies POL6926 will be tested in vivo in animal models of MM to determine the contribution of CXCR7 in EPC trafficking and its contribution to angiogenesis progression in MM. Disclosures: Zimmermann: Polyphor: Employment. Patel:Polyphor: Employment. Romagnoli:Polyphor: Employment. Roccaro:Roche:. Ghobrial:Novartis: Consultancy, 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.

Abstract 2424 Introduction Tumor necrosis factor receptor super families (TNFRSFs) play an important role in activation of lymphocyte and cell apoptosis. However the function of TNFRSFs in multiple myeloma (MM) remains unknown. Loss of function mutation of Fas antigen (TNFRSF6) was identified in MM cells, thus suggesting the possible role of TNFRSFs in regulating MM pathogenesis. We therefore investigated the epigenetic mechanisms that may mediate inactivation of TNFRSFs and its functional role in MM. Methods Dchip software was utilized for analyzing gene expression dataset. DNA was extracted from both primary CD138+ MM plasma cells and MM cell lines using blood & tissue DNA isolation kit (Qiagen, Inc.). Expression of GITR in primary CD138+ plasma cells was detected by Imunohistochemistry (IHC) DNA methylation was analyzed by methylated DNA immunoprecipitation (Medip) assay and bisulfate sequencing. 5'azacytidine was used to demethylate genomic DNA. Gene expression was detected by qRT-PCR and confirmed at the protein level by flow cytometry and western-blot. Over-expression of GITR was obtained in MM1.S cells by using GITR recombinant plasmid and electroporation. Apoptosis was determined using Annexin/PI staining and flow cytometry analysis. Activation of apoptotic signaling was studied by western blot. Cell survival and proliferation were analyzed by MTT and BrdU assay, respectively. Recombinant GITR-lentivirus was obtained from the supernatant of culture medium after 72 hours transfection in 293 cells. GFP positive MM cells were sorted and analyzed by flow cytometry. In vivo effect of GITR on MM tumor growth was determined by injection of GITR over-expressing MM cells in null mice. Mice skull, femur and vertebrae were isolated after 4 weeks injection. Anti-human CD138+ mAb microbead was used to detect MM cells extracted from mice tissue by flow cytometry. Results Gene-expression profiling showed down-regulation of TNFRSFs, including TNFRSF11A, TNFRSF11B, TNFRSF8, TNFRSF10C, TNFRSF9, TNFRSF21, TNFRSF1B, TNFRSF1A and TNFRSF18, compared to normal plasma cells. Moreover, Our IHC results also showed that GITR expression was positive in primary CD138+ plasma cells from 9 normal bone marrow, but negative in 9 MM samples. Importantly, we found that low GITR expression significantly correlated with MM progression. Indeed, GITR gene levels were lower in smoldering and active MM patients compared to MGUS patients and normal donors. Promoter CpG island (CGI) methylation of GITR was indentified in 5 out of 7 MM primary bone marrow (BM)-derived CD138+ cells but not in normal BM-derived plasma cells. Bisulfate sequencing and Medip assay showed that methylation of GITR was significantly associated with GITR expression in 5 MM cell lines, including MM1.S, OPM1, U266, RPMI and INA6. Promoter CGI of GITR was highly methylated leading to complete silencing of GITR in MM1.S cell line. GITR expression was significantly up-regulated in MM cells upon treatment with the 5'azacytidine. MTT and BrdU assay revealed that the proliferation and survival of MM1.S cells was disrupted in the GITR over-expressing MM1.S cells, notably with inhibition of cell proliferation compared to control vector infected cells. Moreover induction of cytotoxicity in GITR over-expressing cells was confirmed by using GFP competition assay. GITR-induced apoptosis was supported by induction of caspase 8 and 3 cleavage. The inhibition of human CD138+ plasma cell growth in the bone marrow of SCID mice using a disseminated MM xenograft model was observed in the experimental group injected with GITR expressing cells compared to the control group after 4 weeks injection. Conclusion Our findings uncovered a novel epigenetic mechanism contributing to MM pathogenesis, showing the role of GITR methylation as a key regulator of MM cell survival. 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.

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 Conclusions. These findings demonstrate the existence of exosome-driven interactions between the BM milieu and MM cells, and suggest that exosomes might constitute a novel mechanism for intercellular transfer of genetic information in the form of miRNAs in clonal plasma cell disorders, such as MM. Disclosures: Roccaro: Roche: Advisory Board. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Ghobrial:Novartis: Advisory Board; Celgene: Advisory Board; Millennium: Advisory Board; Noxxon: Advisory board; Millennium: Research Funding; Bristol-Myers Squibb: Research Funding.

Abstract 2728 Background. We and others have previously demonstrated that primary Waldenstrom's Macroglobulinemia (WM) and Chronic lymphocytic leukemia (CLL) cells show increased expression of microRNA-155 (miR-155), suggesting a role in regulating pathogenesis and tumor progression of these diseases. However, developing therapeutic agents that specifically target miRNAs has been hampered by the lack of appropriate delivery of small RNA inhibitors into tumor cells. We tested the effect of a novel LNA (locked nucleic acid)-modified anti-miR-155 in WM and CLL. Methods. WM and CLL cells, both cell lines (BCWM.1; MEC.1) and primary tumor cells; BCWM.1 Luc+ cells; and primary WM bone marrow (BM) stromal cells were used. WM and CLL cells were treated with antisense LNA anti-miR-155 or LNA scramble oligonucleotide. Efficiency of delivering FAM-labeled LNA into cells was determined by flow cytometry. Survival and cell proliferation were assessed by MTT and thymidine uptake assay, respectively. Synergistic effects of LNA with bortezomib were detected on BCWM.1 or MEC1 cells. Co-culture of BCWM.1 or MEC1 cells with WM bone marrow stromal cells was performed to better define the effect of the LNA-anti-miR155 in the context of the bone marrow microenvironment. miR-155 levels were detected in stromal cells from WM patients by qPCR. Co-culture of BCWM.1 or MEC1 cells with either wild-type or miR155−/− mice BM stromal cells was examined after LNA treatment. Gene expression profiling analysis was performed on BCWM.1 cells treated with either LNA anti-miR-155 or scramble control. miR-155 target gene candidates were predicted by TargetScan software. mRNA levels of miR-155, and its known target genes or gene candidates were detected by qRT-PCR. A microRNA luciferase reporter assay was used to determine whether miR-155 target candidates could be directly regulated by miR-155. mRNA levels of miR-155 targets were detected by qRT-PCR from primary WM or CLL cells treated with LNA. The activity of the LNA-anti-miR-155 was also detected in vivo using bioluminescence imaging and mRNA levels of miR-155 targets were detected by qRT-PCR ex vivo. Efficiency of introducing the FAM-labeled LNA into mice BM cells was determined by flow cytometry 1 week or 2 weeks after intravenous injection. Results. The efficiency of delivering LNA oligos into both WM and CLL-derived cell lines and primary samples was higher than 90%. LNA antimiR-155 reduced proliferation of WM and CLL-derived cell lines by 30–50%, as compared to LNA scramble control. In contrast, LNA antimiR-155 didn't exert significant cytotoxicity in BCWM.1 or MEC.1. LNA synergistically decreased BCWM.1 or MEC1 cell growth co-treated with bortezomib and decreased BCWM.1 or MEC1 cell growth co-cultured with WM BM stromal cells in vitro. A higher level of miR-155 was found in WM BM stromal cells compared to normal ones. LNA decreased BCWM.1 or MEC1 cell growth when co-cultured with BM stromal cells from miR155−/− mice compared with wild-type. We demonstrated increased expression of miR-155-known targeted genes, including CEBPβ, SOCS1, SMAD5, and several novel target candidates including MAFB, SH3PXD2A, and SHANK2, in WM cells upon LNA anti-miR-155 treatment. These target candidates were confirmed to be directly regulated by miR-155 using a luciferase reporter assay. mRNA levels of miR-155 targets were upregulated by 1.5–2 fold at 48 hr after direct incubation of the LNA with primary WM or CLL samples, indicating efficient delivery and biologic effect of the LNA in cells. Moreover, this LNA showed significant in vivo activity by inhibiting WM cell proliferation in a disseminated xenograft mouse model. Upregulation of miR-155 targeted genes were confirmed ex vivo, in WM cells isolated from the BM of treated mice compared to control. Mice BM cells were FAM positive 1 or 2 weeks after injection indicating efficient delivery of FAM-labeled LNA into cells in vivo. Summary. A novel LNA (locked nucleic acid)-modified anti-miR against miR-155 could be highly efficiently delivered into tumor cells in vivo in the bone marrow microenvironment. Anti-WM activity of LNA anti-miR-155 was confirmed both in vitro and in vivo and anti-CLL activity was confirmed in vitro. Novel miR-155 direct target genes including MAFB, SH3PXD2A, and SHANK2 were identified. These findings will help to design individualized clinical trials for WM and CLL patients with elevated levels of miR-155 in their tumor cells. Disclosures: Roccaro: Roche:. Obad:Santaris Pharma: Employment. Broom:Electroporation: Employment. Kauppinen:Santaris Pharma: Employment. Brown:Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding. Ghobrial: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; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.

Abstract 887 Background. Stomal-cell-derived factor 1 (SDF-1) is known to be involved in bone marrow (BM) engrafment for malignant tumor cells, including CXCR4 expressing multiple myeloma (MM) cells. We hypothesized that de-adhesion of MM cells from the surrounding BM milieu through SDF-1 inhibition will enhance MM sensitivity to therapeutic agents. We therefore tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vivo and in vitro. Methods. Bone marrow (BM) co-localization of MM tumor cells with SDF-1 expressing BM niches has been tested in vivo by using immunoimaging and in vivo confocal microscopy. MM.1S/GFP+ cells and AlexaFluor633-conjugated anti-SDF-1 monoclonal antibody were used. Detection of mobilized MM-GFP+ cells ex vivo has been performed by flow cytometry. 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 detection, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12 followed by bortezomib. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–200nM) 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 (combination index: C.I. according to Chou-Talalay method). Results. We first showed that SDF-1 co-localizes in the same bone marrow niches of growth of MM tumor cells in vivo. NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BM stromal cells in vitro. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood (PB) as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the PB of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). 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 (50–100 nM) and bortezomib (2.5–5 nM) was observed (C.I.: all between 0.57 and 0.76). These findings were validated in vivo: tumor burden detected by BLI was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P Conclusion. Our data demonstrate that the SDF-1 inhibiting Spiegelmer NOX-A12 disrupts the interaction of MM cells with the BM milieu both in vitro and in vivo, thus resulting in enhanced sensitivity to bortezomib. Disclosures: Roccaro: Roche:. Kruschinski:Noxxon Pharma AG: Employment. 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; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Advisory Board, Research Funding.

Abstract 1844 Background. We have previously shown the SDF1/CXCR4 axis plays a major role in homing and trafficking of multiple myeloma (MM) to the bone marrow (BM), and disruption of the interaction of tumor cells with the BM leads to enhanced sensitivity to therapeutic agents. We hypothesize that the novel anti-CXCR4 antibody, BMS936564/MDX-1338, may prevent the homing and adhesion of MM cells to the BM and will sensitize them to therapeutic agents. Methods. Primary MM cells (CD138+); MM cell lines (MM.1S, RPMI.8226); and primary MM bone marrow stromal cells (BMSCs) were used. Migration towards SDF-1 and BMSCs has been evaluated. Cytotoxicity and DNA synthesis were measured by MTT and thymidine uptake, respectively. Cell signaling and apoptotic pathways were studied by Western Blot. Synergism was calculated using the Chou-Talalay method. In vivo MM tumor growth was evaluated with xenograft mouse models. Results. MDX-1338 inhibited migration of MM cells toward SDF-1a and primary MM BMSCs, in a dose-dependent manner. Adhesion of primary MM cells to BMSCs was also inhibited by BMS936564/MDX-1338 in a dose-dependent manner, while also inducing cytotoxicity on primary BM-derived CD138+ cells. BMS936564/MDX-1338 targeted MM cells in the context of BM milieu by overcoming BMSC-induced proliferation of tumor cells. In addition, BMS936564/MDX-1338 synergistically enhanced bortezomib-induced cytotoxicity in MM cells. BMS936564/MDX-1338-dependent activation of apoptotic pathways in MM cells was documented, as shown by cleavage of caspase-9 and PARP. SDF-1a-induced ERK-, Akt-, and Src-phosphorilation was inhibited by BMS936564/MDX-1338 in a dose-dependent manner. Importantly, BMS936564/MDX-1338 inhibited MM cell proliferation in vivo in xenograft mouse models. Conclusion. These studies therefore show that targeting CXCR-4 in MM by using BMS936564/MDX-1338 represents a valid therapeutic strategy in this disease. Disclosures: Roccaro: Roche:. Kuhne:BMS: Employment. Pan:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Ghobrial:Noxxon: Research Funding; Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celegene:; Novartis:.

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.

Abstract 4035 INTRODUCTION: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis, implying that progression of MM occurs through continuous interaction between the BM and MM cells, which controls the ability of MM cells to egress out of the BM and home into new BM niches. We have previously shown that the CXCR4/SDF1 axis as well as Rho GTPases downstream of the receptor was important for chemotaxis, adhesion, homing and egress of MM cells. However, the driving force for MM cells to leave the BM and metastasize to other BM sites is not well understood. Regions of severe oxygen deprivation (hypoxia) arise in tumors due to rapid cell division and are associated with poor patient prognosis, cell motility, associated angiogenesis and metastasis. In this study, we tested the role of hypoxia in the dissemination of MM cells in vivo, as well as regulation of the retention/egress of MM cells in and out of the BM. METHODS: To test the effect of hypoxia on induction of MM egress, MM1s-GFP+/Luc+ cells were injected into 12 SCID mice, and then mice with different stages of tumor development (based on the tumor size detected by bioluminescence) were treated with the hypoxia marker pimonidazole. Blood was drawn and BM was obtained from the femur. Mononuclear cells were then fixed, permeabilized, and stained with antibodies against pimonidazole, followed with an APC- secondary antibody, PE-mouse-anti-human CXCR4, and anti-cadherin antibody followed by an Alexa-Fluor-594 secondary antibody. MM cells in BM and peripheral blood were identified by gating on cells with high GFP signal. To confirm the effects of severe hypoxia found in vivo compared to physiologic mild hypoxia found in the BM, we tested the effect of mild hypoxic conditions (6% O2) and severe hypoxic conditions (0.5% O2) on MM expression of cadherins and CXCR4, as well on functional adhesion of MM cells to stromal cells and chemotaxis. RESULTS: Twelve mice with different stages of MM tumor progression were used. A bi-phasic correlation between tumor progression and the percent of hypoxic cells in BM was found, showing that severe hypoxic conditions in the BM correlated with tumor burden. The correlation between the tumor burden and the number of circulating cells was not linear; however, a direct linear correlation was observed between the number of circulating MM cells and hypoxia in the BM. Moreover, hypoxia in BM correlated directly with the expression of CXCR4 and negatively correlated with the expression of cadherins in MM cells isolated from the BM. To test the effect of the severe hypoxic conditions induced by tumor progression compared to mild hypoxic conditions found physiologically in the BM, we tested the effect of 0.5% O2 (severe hypoxia) and 6% O2 (mild hypoxia) compared to normoxia (21%) on MM cell adhesion to BMSCs, as well as on chemotaxis in response to SDF1, as well as expression of CXCR4 and cadherins. We found that severe hypoxic conditions decreased MM expression of cadherins and adhesion to BMSCs, as well as increased expression of CXCR4 and chemotaxis to SDF1 compared to cells in normoxia. In contrast, mild hypoxic conditions did not alter the expression of CXCR4 and cadherins, adhesion of MM cells to BMSCs, or chemotaxis of MM to SDF1 compared to normoxic cells. CONCLUSION: Hypoxia in the BM directly correlates with the number of circulating MM cells, and with changes in expression of cadherins and CXCR4 in vivo. Severe hypoxic conditions, but not mild hypoxic conditions, induce hypoxic responses in MM cells. Based on these findings, further studies to manipulate hypoxia in order to regulate tumor dissemination as a therapeutic strategy in MM are warranted. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. 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.