ALBERT

Description: Multiple myeloma (MM) is characterized by clonal proliferation of transformed plasma cells1 and is extremely dependent on bone marrow (BM) niche molecules and cells, such as osteoclasts. Unlike osteoclasts, the roles of BM adipocytes (BMAs) in MM are poorly understood, despite their great therapeutic potential. This year we published a study of body composition PET/CT parameters that serve as predictors of monoclonal gammopathy of undetermined significance (MGUS) progression to MM(Veld J, O'Donnell EK, Reagan MR, et al. Abdominal adipose tissue in MGUS and multiple myeloma. Skeletal Radiol.). We found that recently diagnosed MM patients had higher abdominal white adipose tissue (WAT) than MGUS patients, even after correction for BMI. Bone Marrow Adipose Tissue (BMAT), a newly appreciated adipose depot with endocrine and paracrine signaling functions, resides near MM cells and has unique expression profiles and phenotypic responses compared to WAT. Because obesity and aging, risk factors for MM, correlate with increased BMAT, and BMAs and MM cells are closely physically associated, we hypothesized that BMAs contribute to an optimal microenvironment for MM cell proliferation and/or drug resistance. We performed direct and indirect co-culture experiments to study the effects of BMAT and BMAT-derived cytokines and lipids on MM proliferation and chemoresistance. MM cells were cultured on, or with conditioned media (CM) from, human and mouse BM-derived mesenchymal stem cells (MSCs) differentiated into adipocytes. MM proliferation, assessed by bioluminescence imaging, was dependent on MM cell line, MSC donor, and adipogenic stage. IL6 is a highly potent MM-supportive cytokine elevated in MM patient BM and thought to be derived mainly from MSCs. MM cells (OPM2 and MM1R) grown in CM from MSCs differentiated for 21 days into adipocytes (Fat CM) treated with IL6 neutralizing antibodies had significantly decreased proliferation vs MM cells treated with Fat CM alone. MM1S cells also showed this trend. These data identified BMAs as a novel BM IL6 source. MM cells typically proliferated in response to donor "lipid fractions", the oil layer on top of human hip surgery BM samples, after 24, 48 and 72 hours, although donor variability was again observed. Lipid droplet content (Oil Red O quantification) of these BMAs also significantly decreased upon culture with MM cells, suggesting that MM cells induce lipolysis or uptake BMAT lipids to fuel their proliferation. In contrast to the literature, we found that adiponectin can be either MM-supportive or MM-inhibitory, depending on the MM line tested and on the presence of dex. Certain MM cell lines (MM1S) became dexamethasone (dex) resistant when treated with Fat CM. Strikingly, all 3 cell lines tested (MM1S, MM1R and OPM2) showed significant decreases in cell number at 24, 48 and 72 hours after treatment with a neutralizing adiponectin antibody vs IgG control, when grown in the presence of 0.1μM dex + Fat CM (which contained high levels of adiponectin from ELISA analysis) (Fig 1A). These data suggest that adiponectin can induce dex resistance, indicating that adiponectin inhibitors + dex may be a novel MM therapy. Lastly, we developed a physiologically relevant 3D in vitro tissue engineered BMAT model utilizing biocompatible, porous silk fibroin scaffolds to more accurately define BMA-MM interactions. Our 3D models provide the correct mechanical robustness and biomaterial properties to mimic trabecular bone and unilocular BMAT (Fig 1 B-D). We generated long-term cultures of BMAT from MSCs and cultured MM cells (GFP+ MM1S) on these for up to 1 week, demonstrating the development of the first 3D BMAT artificial culture system, with or without MM cells. We are now using this novel platform to more deeply explore the relationship between BMAT and MM cells. In conclusion, BMAT likely plays a role in MM progression. 3D tissue engineered models of the BM milieu are a crucial link between 2D and in vivo models, maintaining the high-throughput capacity of 2D studies and the translational relevancy of in vivo models. Our data demonstrate important interactions between BMAT and MM cells, highlighting our need for further research into the roles of BM adipokines and adipocytes in MM pathogenesis and chemoresistance. Disclosures No relevant conflicts of interest to declare.

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: Key Points Knockdown of the sialyl-transferase, ST3GAL6, in MM inhibits in vivo homing and prolongs survival in xenograft mice. In MM patients, high expression of ST3GAL6 is associated with inferior overall survival.

Description: Background. LIN28B regulates developmental processes and cellular reprogramming by suppressing let-7 microRNAs (miRNAs). A role for LIN28B has been reported in cancers, however the LIN28B/let-7 axis has not been studied in multiple myeloma (MM). Methods. LIN28B level expression in MM patients was studied using previously published gene expression profiling (GEP) datasets. Knockdown (KD) of LIN28B was performed on MM cell lines (U266, MOLP-8) using 2 shRNA and validated using 2 sgRNA by CRISPR knockout (KO). Downstream regulations were assessed by qRT-PCR and western blots, as well as RNA sequencing. For RNA sequencing, control and Lin28B CRISPR cells were used for library preparation (NEBNext kit) and sequencing on a HiSeq 2000. Proliferation of KD and KO cells were evaluated in vitro and in vivo in a xenograft mouse model. An LNA-GapmeR technology was used to develop a let-7 mimic in vivo in SCID mice. Findings. Two independent GEP datasets (GSE16558; GSE2658) were analyzed for LIN28B expression, showing a significantly higher level in MM patients compared to healthy controls. In addition, high LIN28B levels correlated with a shorter overall survival (p = 0.0226), along with an enrichment of let-7 target genes by Gene Set Enrichment Analyses (GSEA). LIN28B KD cells had a significantly increased expression level of let-7 family members and were associated with down-regulation of let-7 target genes Myc and Ras at the protein level. We further confirmed downstream regulation of MYC and RAS in a LIN28B CRISPR KO model in MM cells (MOPL-8). We next validated the role of LIN28B in MM in vivo by using a xenograft tumor model showing a decreased tumor burden in LIN28B KD mice compared to scramble control (p =0.0045). In addition, we performed a RNA sequencing from the CRISPR LIN28B KO and control cells and observed a central role by GSEA for both MYC and E2F cell cycle pathways in LIN28B-engineered cells. LIN28B activity in regulating MYC and cell proliferation was further defined to be dependent on let-7 by performing a rescue experiment in MM1S cells. Moreover, we explored the possibility to therapeutically regulate MYC expression through let-7 with an LNA-GapmeR containing a let-7b mimic, in vivo, and showed that high levels of let-7 expression represses tumor growth in SCID mice by regulating MYC expression compared to control GapmeR treated mice (p = 0.0026). Conclusions. These findings reveal the essential role of LIN28B/let-7 in regulating two essential oncogenic pathways in MM, MYC and RAS. Interference with this pathway may represent an efficient option for targeting MYC in cancer. Disclosures No relevant conflicts of interest to declare.

Description: Background. p53 is a well defined tumor suppressor involved in the modulation of cell proliferation, cell cycle progression and programmed cell death. BLIMP-1 plays a crucial role in modulating B-cell differentiation towards Ig-secreting plasma cells, and it acts as a tumor suppressor, as documented in both diffuse large B-cell lymphoma and Burkitt lymphoma. Whether B-cell specific loss of both p53 and BLIMP-1 may favor a B-cell lymphoma phenotype remains unanswered. We therefore aimed to generate in vivo dual p53/BLIMP-1-floxed conditional inactivation in B-cells, and to define the functional relevance of both p53 and BLIMP-1 n B-cell lymphomagenesis in vivo Methods.Cre recombinase under the control of CD19 promoter (C57BL/6 CD19Cre/Cre) mice were crossed with either C57BL/6 BLIMPflox/flox or C57BL/6 p53flox/flox mice to achieve deletion of BLIMP or p53, respectively, in B cells. Secondly, CD19Cre/Cre BLIMPflox/flox mice were crossed with CD19Cre/Cre p53flox/flox to achieve concomitant deletion of both BLIMP and p53 in B cells (CD19Cre/Cre BLIMPflox/flox p53flox/flox), referred as CD19/Bl-/p53- mice. Transgenic experimental mice (CD19/Bl-/p53-) where characterized for B cell infiltration using immunohistochemistry, flow cytometry; clonotypic immunoglobulin heavy-chain rearrangement was assessed by Southern Blotting. Whole exome sequencing was performed using DNA isolated from B220+ selected cells obtained from pathological lymph nodes of CD19/Bl-/p53- mice and from matched tail-derived tissues, used as germline (Illumina HiSeq 2500 platform; Agilent SureSelectXT). MTT assay was used to BTK-inhibitor-dependent cytotoxicity using CD19/Bl-/p53-derived B220 cells. Results.We generated dual p53/BLIMP-1-floxed conditional inactivation in B-cells, using mice expressing Cre recombinase under the control of CD19 promoter. 100% of the CD19/Bl-/p53- mice presented with diffuse lymphadenomegalies, and splenomegaly, hepatomegaly (90.3% and 77.4%, respectively). Other clinical manifestations included presence of ascites and hind lymb paralysis (12.9% and 19.3%, respectively). The CD19/Bl-/p53- showed worse survival compared to Bl-/p53- mice non-expressing the CD19/Cre recombinase, CD19/p53-, or CD19/Bl- (363, 469.5, 460.5, and 770 days, respectively). H.E. staining of CD19/Bl-/p53--derived lymph nodes, defined a nodal architecture with a monomorphic population of large sized atypical lymphoid cells with finely clumped and dispersed chromatin, and multiple basophilic medium sized, paracentrally situated nucleoli. A "starry sky" pattern was also observed. Overall, these features are compatible with a high-grade lymphomas. IHC analysis confirmed a marked positivity for B220 staining (TdT, Bcl6, CD138 and CD4, CD8 negative). Tumors were confirmed to be B220+/IgM+, with either Igk- or Ig-lambda-restriction as demonstrated by flow cytometry; and either mono- or bi-clonal, as demonstrated by Southern blotting, thus further confirming the clonal transformation induced by dual BLIMP/p53 deletion in B cells. Whole exome sequencing was performed from B220+ selected cells obtained from pathological lymph nodes of CD19/Bl-/p53- mice and identified 143 SNVs. Among them, non-synonymous somatic mutations were mapped on genes involved in the regulation of focal adhesion, PDGF signaling, p53-downstream pathway, and lipoprotein metabolism. B220+ cells selected from CD19/Bl-/p53--derived lymph nodes were implanted subcutaneously into recipient SCID/Bg mice (n: 10), and presented with 100% engraftment, with a monomorphic lymphoid infiltration of B220+ and IgM+ cells. B220 positive cells were selected from the s.q. tumor and intravenous injected into recipient SCID/Bg (n: 10) and BL/6 mice (n: 10). Engraftment was demonstrated in all the mice, where hepatomegaly, splenomegaly and hind lymb paralysis were observed. Infiltration of B220+ cells was documented within bone marrow, liver and spleen. We next investigated the anti-tumor activity of BTK-inhibitor, and found that B220+ cells selected from lymph nodes harvested from CD19/Bl-/p53-mice were sensitive to ibrutinib treatment. Conclusion. These studies demonstrate that the specific dual inactivation of p53 and BLIMP in B-cells promotes oncogenic transformation, resulting in aggressive B-cell lymphoma development. Disclosures Ghobrial: Celgene: Other: Advisory Board; BMS: Other: Advisory Board; Amgen: Other: Advisory Board; Takeda: Other: Advisory Board; Janssen: Other: Advisory Board. Roccaro:Takeda Pharmaceutical Company Limited: Honoraria.

Description: Abstract 1837 Introduction: Current research has demonstrated that cancer cells are not only influenced by their microenvironment, but are also able to drastically alter their surroundings to further accelerate cancer progression. Hematological malignancies create a forward feedback system with local mesenchymal stromal cells within the bone marrow, but the exact mechanisms and cellular changes within the stroma are largely unknown. Our work has aimed at understanding the bidirectional interaction between multiple myeloma (MM) cells and human bone marrow-derived mesenchymal stromal cells (hMSCs) by characterizing hMSCs derived from either MM patients or healthy individuals, using medium-throughput assays and 2D and 3D in vitro bone marrow models. Methods: First, primary human myeloma patient MSCs (MM-MSCs) and normal donor MSCs (ND-MSCs) were characterized in terms of proliferation when cultured with and without MM.1S cancer cells in direct and indirect co-culture. Next, primary ND- and MM-MSCs were profiled for their microRNA (miRNA) (n=3 for normal, n=7 for MM) and mRNA (n=5 for normal, n=5 for MM) expression using Nanostring technologies. We analyzed 800 human miRNAs from miRBase v.18 and 230 human cancer-related genes using the nCounter® Human Cancer Reference Kit, which allowed for much greater specificity and reliability than microarray technologies. Lastly, a more representative culture system was designed to better mimic myeloma growth within the bone marrow. We developed a 3D in vitro model using hMSCS and fluorescent, luciferase-labeled MM cell lines seeded into porous, autofluorescent silk scaffolds. Scaffolds were made using silk fibroin protein isolated from silkworm cocoons and were formed into biocompatible cylinders with pores of 500–600 microns in diameter. Both ND- and MM-MSCs were labeled with fluorescent cell-tracker dyes and cultured on scaffolds with or without MM1S-GFP+/Luc+ cells. These were non-destructively assessed using confocal microscopy and bioluminescent imaging (BLI) for their ability to promote cancer cell growth and protect cancer cells from chemotherapeutics. Comparison with a conventional 2D in vitro model was performed. Scaffolds were also assessed for their ability to support in vitro culture of primary MM cells using confocal microscopy. Results: MM-MSCs differed from ND-MSCs at miRNA, mRNA, and functional levels. MM-MSCs proliferated slower than ND-MSCs and direct, but not indirect, co-culture of ND-MSCs with MM.1S was able to recapitulate this slowed proliferation in vitro. Indeed, we found that 22 microRNAs were significantly dis-regulated (14 up-regulated and 8 down-regulated in MM patients versus healthy individuals). These included up-regulation of miRNA-222, -181, -146, and -382; together with down-regulation of miRNA-15a, -143, and -199a, in MM-derived hMSCs, compared to ND-MSCs (p

Description: Multiple myeloma (MM) is a malignancy of plasma cells and is characterized by unrestricted tumor cell growth in bone marrow (BM). MM causes destructive osteolytic lesions causing bone fracture, bone pain, hypercalcaemia, and nerve-compression, resulting from increased bone resorption and suppressed bone formation. Despite the introduction of agents to inhibit bone resorption, such as bisphosphonates, which prevent further bone loss, approaches to preventing osteoblast suppression and repair bone lesions are limited and there are no agents available clinically. The wnt/β-catenin pathway plays a critical role in the regulation of bone formation. Production of the soluble wnt antagonist dickkopf1 (Dkk1) by MM cells has been implicated in MM inhibition of bone formation. As such, Anti-Dkk1 treatment prevents bone disease in pre-clinical models of MM and is in early clinical development. However, Dkk1 is not expressed by all myeloma cells; hence only a proportion of patients may respond to anti-Dkk1 therapy. Sclerostin (Scl) is a soluble wnt antagonist whose expression, unlike Dkk1, is restricted to osteocytes; therefore Scl targeted agents may have less off target effects. Anti-Sclerostin (Anti-Scl) treatment increased bone formation and bone volume in experimental models of osteoporosis, and increased bone mineral density in phase II osteoporosis clinical trials. However, Anti-Scl treatment effects on myeloma bone disease are unknown. Further, cells of the BM such as osteoblasts have been implicated in the regulation of MM cell survival and growth. Thus, in the present study we explored the potential for Anti-Scl therapy to prevent MM induced bone loss and inhibit MM growth in both murine and human xenograft MM models. Female C57BLKalwRij mice (n=8) were injected i.v, with 5TGM1/eGFP murine MM cells (1×106) and female SCID/beige mice (n=10) were injected i.v. with MM1S/Luc/eGFP human MM cells (4 × 106). 24 hours later, naïve mice (without tumor cells) or mice bearing MM cells were treated with anti-sclerostin antibody (Anti-Scl) (100mg/kg i.v) or control antibody. Mice were sacrificed at day 21 (MM1S) or day 28 (5TGM1) and the effect of Anti-Scl on bone structure in the femora and vertebrae were determined by microCT analysis. The effect of Anti-Scl on MM burden was determined by bioluminescent imaging (BLI) performed twice weekly from week 1 using a Xenogen IVIS system, whereas MM burden in 5TGM1/eGFP bearing mice was examined by FACS analysis. Anti-Scl treatment in naïve C57BLKalwRij mice increased trabecular bone volume fraction (BV/TV, 39%, p

Description: Background Extramedullary disease (EMD) in patients with multiple myeloma occurs mostly in advanced disease or relapse. EMD seems to have a different pathogenesis from medullary myeloma and is often characterized by a more aggressive clinical course. To date molecular mechanisms of development of EMD have not been fully understood. Methods: Human MM cell lines, IM-9 and MM1S were serially selected in immune-deficient mice. IM-9 and MM1S cells were inoculated intravenously and harvested from the tumors developed in the bone marrow (BM) and liver (site of extramedullary disease), aiming to establish BM-prone and liver-prone clones. Tumor progression was periodically checked by bioluminescence (BLI) and in vivo live confocal imaging. After three rounds of in vivo selections, the cells of both BM- and liver-prone were characterized by gene and protein expression and cellular functional assays. Results: We obtained three liver-prone sub-clones in both IM-9 and MM1S after serial in vivo selections. These cells had equal proliferation rates in vitro compared to the original or BM-prone cells, but exhibited more aggressive phenotype in vivo. Liver-prone clones had significantly higher migration ability than BM-prone clones (11.6% vs 6.1% migration, respectively p=0.018). Gene and protein expression analysis revealed that each liver-prone clone had a higher expression of sets of chemokine receptors specifically CXCR4. Using thein vivo metastatic model CXCR4-over expressing myeloma cells exhibited higher metastatic property to the extramedullary organs whereas CXCR4-knockdown cells has less tumor metastasis to the liver. Discussion: We established EMD-prone human multiple myeloma cell lines that reproducibly developed liverinvolvement consistent with human extramedullary disease. These cells exhibited higher migratory ability and increased expression of several chemokine receptors, specifically CXCR4. We validated the effect of CXCR4 on developing extramedullary myeloma using our established in vivo mode. Further studies to determine the role of CXCR4 for therapeutic targeting of extramedullary disease in MM are ongoing. Disclosures: Ghobrial: Sanofi: Research Funding; Noxxon: Research Funding; BMS: Advisory board, Advisory board Other, Research Funding; Onyx: Advisoryboard Other.

Description: Background. Endothelial progenitor cells (EPCs) are circulating precursors with the capacity to differentiate into endothelial lineage cells through a process known as “vasculogenesis” thus contributing to vessel formation. We studied the role of endothelial progenitor cells in multiple myeloma (MM) pathogenesis. Methods. EPC levels were evaluated in peripheral blood (PB) of patients with smoldering MM (SMM), and active myeloma (MM) and in PB of healthy controls, by using flow-cytometry (CD34+VEGFR2+ cells), and by performing endothelial colony forming assays. EPC levels were studied in PB from transgenic Vk*MYC mice harboring either early MM (smoldering-like stage); or late (active MM-like stage); as well as in mice injected intravenously with either murine MM 5TGM1-turboRFP+ cells or human MM1S-GFP+/luc+ cells. Healthy syngeneic mice were used as controls. GFP-bone marrow (BM) transplantation and sub-cutaneous femur implantation were performed in recipient mice to study EPC BM mobilization (GFP+CD34+VEGFR2 cells) and EPC BM recruitment to the implanted femurs during 5TGM1 MM model progression. Transgenic ID1+/-ID3-/- mice (with a defect in EPC mobilization and differentiation ability) were injected with transplantable Vk*MYC cells and followed for survival. Wild type littermates were used as controls. Similar experiments were performed using transgenic ID1+/-ID3-/- mice transplanted with BM of wild type littermates. Therapeutic activity of DC101 anti-murine VEGFR2 Ab was evaluated in the MM1S human orthotopic xenograft model, using both early and late treatment approaches. Results. EPC levels were significantly increased in PB of MM patients, including SMM, compared to healthy controls (P

Description: Background The extracellular matrix (ECM) is a major component of the tumor microenvironment, contributing to the regulation of cell survival, proliferation, differentiation and metastasis. In multiple myeloma (MM), interactions between MM cells and the bone marrow (BM) microenvironment, of which the ECM forms a major component, are critical to the pathogenesis of the disease and the development of drug resistance. To date, despite some knowledge of the composition of the ECM in tumors, detailed profiling of the composition of the ECM in MM has not been carried out. Until recently ECM proteins have proven difficult to characterize due to their biochemical properties and large size. Recent advances in proteomics have led to the characterization of the ECM and ECM-associated proteins (“matrisome”) in normal human tissues and tumors using a systematic and comprehensive approach. Methods Tumor Xenograft models; MM1S-GFP-Luc+ cells (5x106) were injected intravenously into SCID-Bg mice (n=4/group) and animals underwent weekly bioluminescent imaging (BLI). Mice were sacrificed after 2 weeks in order to mimic early tumor development (luminescence = 1x105 p/sec/cm2/sr) or 5 weeks (1x108 p/sec/cm2/sr) to model more advanced MM. Human bone marrow aspirates; Whole bone marrow was obtained from newly diagnosed MM patients (n=9) and healthy human donors (ND) (n=4) following written informed consent. ECM proteins were enriched from bone marrow samples obtained from MM patients, NDs and mice according to previously published methods.Tandem Mass Spectrometry (LC-MS/MS): Peptides were run using reversed-phase microcapillary liquid chromatography – tandem mass spectrometry (LC-MS/MS) on a high resolution hybrid Orbitrap Elite mass spectrometer. MS/MS data were searched against the UniProt Human database using MASCOT to identify proteins. Spectral counts were used as a semi-quantitative measure of abundance. ECM proteins were defined according to the in-silico definition of the matrisome. Validation of expression of ECM mRNA in MM cell lines (MM1s, RPMI-8226 and U266) and in CD138+ cells and bone marrow stromal cells (BMSC’s) from MM patients in comparison to NDs was performed using qRT-PCR. Results Primary myeloma sample ECM; Using a spectral count of 2 as a cutoff of peptide abundance we identified a total of 536 unique proteins in ND bone marrow of which 35 are defined as matrisome proteins. 982 unique proteins were enriched from whole bone marrow samples of newly diagnosed MM patients of which 26 are defined as matrisome proteins, 7 unique proteins were identified as ECM or ECM-associated in newly diagnosed patients which were not detected in the ND samples including PRG3, FGG, LEG10, TLN1 and PLEC. Critical ECM components such as laminins, matrix metalloproteinases and collagens were also found to be significantly altered in newly diagnosed MM with evidence of destruction of ECM components in active disease. Tumor Xenograft ECM; In mice with an earlier phase of human MM1s cell tumor burden we detected a total of 329 unique proteins of which 48 were defined as matrisome proteins, 23 of these proteins were unique to the earlier phase of MM in these mice. Mice with more advanced tumor development had unique ECM proteins which were not detected in the earlier disease stage including collagens, laminins and matrix metalloproteinases, indicating that these ECM components may be critical for re-modelling the ECM in MM. Interestingly, in our xenograft model of MM we were able to detect both human and mouse ECM components indicating that the tumor ECM is secreted from both the murine stroma and the human MM cells and allowing delineation of the source of individual ECM components. This indicates that as MM progresses certain ECM components, including FBN1, which were initially derived from stroma are later derived from MM cells. Differential expression of ECM components, including FBN1 between normal and malignant plasma cells was confirmed using qRT-PCR. Conclusions We have performed proteomic profiling of the unique tumor ECM in MM using mass spectrometry with a view to determining the specific components that may be altered with disease progression. Through this approach plasma-cell-derived ECM can be identified with a view to developing therapeutic strategies in this disease. Disclosures Glavey: BMS: Consultancy, Research Funding. Palumbo:Bristol-Myers Squibb: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria. Ghobrial:Onyx: Advisory board Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.