ALBERT

Description: Abstract 2920 Background: Nicotinamide adenine dinucleotide (NAD+) is a coenzyme crucially involved in several cellular functions, including energy metabolism, reactive oxygen species scavenging, DNA repair, and gene expression. Intracellular NAD+ stores are continuously replenished through pathways whose activity depends on the tissue and availability of substrates. Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme in the NAD+ salvage pathway from nicotinamide. During neoplastic transformation, Nampt is upregulated to compensate for increased metabolic demands. It promotes myeloid and lymphoid differentiation and increases specific cytokine production (TNF- α, IL-6 and VEGF). Importantly, cancer and leukaemia cells appear to be more sensitive to Nampt inhibitor drugs than normal cells. The reasons for this selectivity are not fully understood, but may include aberrant metabolic demands and increased reliance on NAD+-dependent enzymes. Promising results obtained with Nampt inhibitors (such as FK866) in preclinical cancer models suggest that Nampt activity represents an innovative therapeutic target for novel anticancer agents. Methods: A panel of eighteen different MM cell lines, both sensitive and resistant to conventional and novel anti-myeloma drugs, as well as patient MM cells were used in the study. The mechanism of action of FK866 was investigated by Annexin-V/propidium iodide staining, thymidine incorporation, Western-blotting, and with lentivirus-mediated shRNAs. For the autophagy assay, EGFP-LC3+ cells were treated with FK866 and the number of GFP-LC3 punctae was analyzed and quantified by fluorescence microscopy and flow cytometry, respectivley. Intracellular NAD+ content was measured using a biochemical assay. Angiogenesis was measured in vitro using Matrigel capillary-like tube structure formation assay. Results: To study the role of Nampt in MM cells, we performed a protein analysis of this enzyme in eighteen MM cell lines. Nampt is constitutively activated in all cell lines tested. Moreover, patient MM cells highly express this enzyme whereas normal cells lack this protein. Indeed, the Nampt inhibitor FK866 decreased MM cell line viability in a dose and time dependent manner, with an IC50 ranging from 3–30nM. Similar results were observed in patient MM cells. Importantly, FK866 did not inhibit viability of normal peripheral blood mononuclear cells. Tritiated thymidine uptake assay confirmed the antiproliferative effects of FK866 in MM cell lines and patient cells. To examine the mechansim of action, we showed that intracellular NAD+ levels decreased with FK866 treatment at 24 and 48 hours. Furthermore, knock-down of Nampt by small interfering RNAs caused significant inhibition of MM cell growth. FK866 triggered anti-MM activity in our models of MM in the bone marrow (BM) microenvironment, confirming its ability to overcome the proliferative advantage conferred by the BM milieu. FK866 treatment also inhibited angiogenesis via suppression of pivotal MM pathways PI3K/AKT and ERK. In further studies to delineate its mechanisms of action, no activation of apoptosis was observed in treated-cells. Instead FK866 treatment resulted in a marked increase in autophagy, evidenced by autophagic vacuoles in the cytoplasm and proteolitic processing of endogenous LC3-I to LC3-II. FK866 inhibited mTOR signaling and triggered increased formation of EGFP-LC3 punctae, confirming involvement of autophagic cell death. Finally, combined FK866 with bortezomib (CI 〈 0.6), melphalan (CI 〈 0.9), and dexamethasone (CI 〈 0.8), induced synergistic cytotoxicity against MM cells. Conclusion: Our data therefore show a pivotal role of Nampt in MM cell growth, survival and drug resistance. The ability of FK866 to inhibit Nampt activity strongly supports its clinical evaluation to improve patient outcome in MM. Disclosures: Hideshima: Acetylon: Consultancy. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 883 Specific expression of Bruton's tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in regulating osteoclastogenesis. Although Btk is critical in B cell maturation and myeloid function, it has not been characterized in plasma cell malignancies including multiple myeloma (MM) and Waldenström Macroglobulinemia (WM). We here investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) microenvironment, as well as on MM and WM cancer cells. We further define molecular targets of Btk signaling cascade in OCs and MM in the BM milieu. In CD14+ OC precursor cells, RANKL and M-CSF stimulate phosphorylation of Btk in a time-dependent fashion; conversely, PCI-32765 abrogates RANKL/M-CSF-induced activation of Btk and downstream PLCγ2. Importantly, PCI-32765 decreased number of multinucleated OC (〉3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and the secretion of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It increased size of OCs and number of nuclei per OC, with significantly defective bone resorption activity as evidenced by diminished pit formation on dentine slices. Moreover, lack of effect of Dexamethasone on OC activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 significantly reduced cytokine and chemokine secretion from OC cultures, including MIP1α, MIP1β, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1–0.48 nM). It potently decreased IL-6, SDF-1, MIP1α, MIP1β, and M-CSF in CD138-negative cell cultures from active MM patients, associated with decreased TRAP staining in a dose-dependent manner. In MM and WM cells, immunoblotting analysis confirmed a higher Btk expression in CD138+ cells from majority of MM patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated a higher expression of Btk and its downstream signaling components in WM cells than in CD19+ normal bone marrow cells. PCI-32765 significantly inhibits SDF-1-induced adhesion and migration of MM cells. It further blocked cytokine expression (MIP1a, MIP-1β) at mRNA level in MM and WM tumor cells, correlated with inhibition of Btk-mediated pPLCγ2, pERK and NF-kB activation. Importantly, PCI-32765 inhibited growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs in IL-6-dependent INA6 and ANBL6 MM cells. Furthermore, myeloma stem-like cells express Btk and PCI-32765 (10–100 nM) blocks their abilities to form colonies from MM patients (n=5). In contrast, PCI-32765 has no adverse effects on Btk-negative BMSCs and OBs, as well as Btk-expressing dendritic cells. Finally, oral administration of PCI-32765 (12 mg/kg) in mice significantly suppresses MM cell growth (p〈 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results provide compelling evidence to target Btk in the BM microenvironment against MM and WM., strongly supporting clinical trials of PCI-32765 to improve patient outcome in MM and WM. Disclosures: Chang: Pharmacyclics Inc: Employment. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership. Elias:Pharmacyclics Inc: Consultancy. Treon:Millennium: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 2913 CRM1 (chromosomal region maintenance 1, XPO1) is the major export protein regulating degradation of key tumor suppressors by transporting them from nucleus to cytoplasm, thus abrogating their function. Highly expressed CRM1 is associated with poor prognosis in several solid tumors, and inhibition of CRM1 restores function of tumor suppressors such as p53, p21 and FOXO and IκB (cellular antagonist of NF-κB). Furthermore, CRM1 knockdown enhances sensitivity of human multiple myeloma (MM) cell lines to topoisomerase II inhibitor (Cancer Res 2009 :69, 17). In this study, we investigated a novel selective CRM1 inhibitor, KPT-185, in human MM cells. Gene expression profiling analysis using GEO database showed that CRM1 expression is significantly increased in CD138+ cells from MM patients versus monoclonal gammopathy of undetermined significance (MGUS) patients or normal donors (p

Description: Abstract 2674 Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and progression of many types of cancer, and VEGF expression has been known to be associated with polymorphism of gene. However, the impact of polymorphisms of the VEGF gene on non-Hodgkin lymphoma (NHL) prognosis has not been fully elucidated. Here we investigated the association between VEGF polymorphisms and prognosis of NHL. The study involved 96 NHL patients treated at National Cancer Center, Korea. The median patient age was 57 years, and 60 patients (62.5%) were men. A total of 5 polymorphisms with heterozygous allele were analyzed. Clinical characteristics (e.g., cell lineage) and international prognostic index (IPI), including age, performance, LDH, stage and extra-nodal involvement, were evaluated and related to VEGF polymorphism. Hazard ratios (HRs) were determined in terms of risk for overall survival using Cox proportional hazard regression analysis. Diffuse large B cell lymphoma (n=73) was most common histologic type, and others were as follows: T-cell lymphoma (n=14), mantle cell lymphoma (n=6), and Burkitt lymphoma (n=3). IPI and stage were predictors for prognosis (p

Description: Malignant cells have a higher nicotinamide adenine dinucleotide (NAD+) turnover rate than normal cells, making this biosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD+ synthesis, Nampt, in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 triggered cytotoxicity in MM cell lines and patient MM cells, but not normal donor as well as MM patients PBMCs. Importantly, FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6, IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD+ stores. Interestingly, cytotoxicity of FK866 triggered autophagy, but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity. Finally, FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model, associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Our data therefore define a key role of Nampt in MM biology, providing the basis for a novel targeted therapeutic approach.

Description: Abstract 4064 HM1.24, an immunological target highly expressed on majority of multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). Recently, XmAb5592, a novel Fc-domain engineered humanized anti-HM1.24 mAb with specific Fc-domain modification, was shown to induce 〉10-fold antibody-dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis against MM cells, when compared with the humanized normal anti-HM1.24 IgG1 (XmAb5627) from which it is derived (ASH Abstract#609, 2009). Here we investigated whether XmAb5592, when combined with other anti-MM drugs, further enhanced ADCC against MM cell lines and primary patient MM cells using Calcein-AM release ADCC assays and flow cytometric analysis for cell membrane CD107a to specifically quantitate NK cell (CD56+CD3-) activation. Addition of lenalidomide (10 μ M) in standard ADCC assays increased XmAb5592 (0.1 μ g/ml)-induced cell lysis against MM1S, MM1R, and RPMI8226 cells in the presence of peripheral blood mononuclear cells (PBMCs) from normal donors (n=2). Specifically, XmAb5592 (0.01, and 0.1 μ g/ml) combined with lenalidomide (2.5, 5, 10 μ M), in the presence or absence of IL-2 (100 units/ml), synergistically induced NK-mediated RPMI8226 MM cell lysis, as evidenced by combination index (CI) 〈 1 (0.08-0.89). Pre-incubation of PBMCs with additional IL-2 (100 units/ml) enhanced even stronger XmAb5592-induced cytotoxicity against MM cells than pretreatment with lenalidomide alone. Lenalidomide also enhanced PBMC effectors to kill primary patient MM cells. Cell surface CD107a, as a functional marker for NK cell activation dependent on granzyme B secretion, was further determined following target myeloma cell incubation, with or without NK cells and in the presence of mAbs. NK cells were activated by as low as 0.001 μ g/ml of XmAb5592 only in the presence of MM1S tumor cells. In contrast, neither XmAb6166, an Fc-domain knockout of XmAb5592 without NK binding, nor XmAb4614, a similar Fc-engineered mAb targeting respiratory syncytial virus (RSV) antigen, induced any cell surface CD107a on NK cells incubated with MM cells. Specific XmAb5592-induced CD107a-dependent NK cell degranulation further correlated with IFNγ secretion (r=0.7, p=0.03). XmAb5592 induced 〉 10-fold more potent NK degranulation than XmAb5627, which significantly correlated with MM cell lysis by ADCC, even in the presence of bone marrow stromal cells (BMSCs). Moreover, no significant HM1.24 was expressed on NK cells stimulated with or without IFN-α, suggesting minimal NK toxicity. XmAb5592 more potently (〉10-fold) than XmAb5627 induces homotypic aggregation and adhesion of NK cells, which was further enhanced by lenalidomide. Finally, minimal HM1.24 expression was confirmed on different PBMC subsets including CD15+ (PMN), CD19+ (B), CD14+ (MC), and CD3+ (T) cells. These results indicate that lenalidomide further potentiates XmAb5592-induced myeloma cell killing via NK-mediated ADCC, providing a rationale to combine both novel drugs to improve patient outcome in MM. Disclosures: Muchhal: Xencor Inc: Employment. Desjarlais:Xencor Inc: Employment. Richardson:Gentium: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Abstract 3013 Daratumumab is a novel fully human therapeutic CD38-specific monoclonal antibody (mAb) that is currently in phase I/II safety and dose finding clinical studies in MM. We recently demonstrated that daratumumab induces antibody dependent cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) against multiple myeloma (MM) cells (ASH Abstract #608, 2009). Significantly, daratumumab induces ADCC-mediated autologous lysis against MM patient cells. In addition, when cross-linked, daratumumab directly induces Ramos lymphoma cell death. We here studied whether daratumumab directly kills MM cells and whether daratumumab could be combined with other anti-MM drugs to further enhance its direct cytotoxicity. Direct daratumumab-induced MM cell death was determined using CellTiter-Glo luminescent cell viability assay and Annexin V/PI flow cytometry analysis, with or without goat anti-human IgG crosslinking. Following 48h incubation, daratumumab (0.1-10 μg/ml), when cross-linked, directly induced cytoxicity against dexamethasone (dex)-sensitive MM1S and dex-resistant MM1R cells, as evidenced by decreased cell viability in a dose-dependent manner. Importantly, cross-linked daratumumab increased caspase 3/7 activities in a dose-dependent fashion, as assessed by the Caspase-Glo® 3/7 luminescence assay. Furthermore, daratumumab upregulated Annexin V+ and Annexin V+/PI+ cells in freshly isolated CD138+ MM patient cells, from 7.7% to 20.6% and 10.9% to 15.4 %. Therefore, cross-linked daratumumab can directly trigger apoptosis of patient myeloma cells. Cell viability assay was further performed on MM1S cells when daratumumab (0.1, 1, 10 μg/ml) was combined with dex (0.5 and 1 μM) or bortezomib (2.5, 5, and 10 nM). Following 48–72h incubation with daratumumab, both dex and bortezomib synergistically inhibited MM cell viability, as determined by combination index (CI) 〈 0.5 at given combined concentrations of these drugs. Enhanced caspase 3/7 activation was also seen when daratumumab was combined with dex. To evaluate combination cytotoxicity induced by lenalidomide with daratumumab, peripheral blood mononuclear effector cells (PBMCs) from normal donors (n=2) were pretreated with lenalidomide (2 μM) for 3 days followed by daratumumab-mediated ADCC assays against MM1S cells. Using calcein-AM release measurements, lenalidomide-pretreated PBMCs further augmented daratumumab-induced MM1S cell lysis, whereas daratumumab-pretreated PBMCs did not alter ADCC. Taken together, our studies show that daratumumab directly induces MM cell death via activation of caspase 3/7 and daratumumab induced synergistic cytotoxicity with dex or bortezomib. Moreover, lenalidomide augments daratumumab-induced ADCC against MM cells. These results further support combination clinical trials of conventional and novel anti-MM drugs with daratumumab in MM. Disclosures: Weers: Genmab: Employment. Parren:Genmab: Employment. Richardson:Keryx Biopharmaceuticals: Honoraria. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Introduction: Recent studies have elucidated the importance of using 3-dimensional rather than 2-dimensional models in order to create an experimental system recapitulating the specialized properties of the bone marrow microenvironment. Since the neoplastic bone marrow (BM) milieu plays important roles in multiple myeloma (MM) pathogenesis, novel models to study the MM cell in its neoplastic microenvironment are needed. Methods: To mimic the neoplastic BM microenvironment of MM patients, we have established a special hydrogel-based 3-dimensional (3-D) model by ex-vivo culturing MM patient-derived mesenchymal stem cells (MM-MSCs), the predominant cellular component of the marrow niche, which promotes greater mineralization and differentiation than a 2-dimensional (2-D) system. Results: To characterize MM-MSCs in different stages of MM, we utilized an 11 multi-color flow cytometry panel. The percentage of MSCs (CD73+CD90+CD105+lin-CD45-CD34-HLA-DR-) population in BM aspirate samples of 50 MM patients (MGUS, smoldering MM, newly diagnosed MM, and relapsed or relapsed/refractory MM) was evaluated, and correlated with the distribution of (CD38+ CD138+) plasma cells. MSCs were less frequent (10x) than plasma cells, and increased with disease progression to relapsed/refractory MM. We seeded MM-MSCs (N=34) which had been expanded by adhesion methods in 2-D versus 3-D models in order to create an ex-vivo MM niche-like structure. In the hydrogel-based 3-D model, MM-MSCs formed compact clusters with active fibrous connections and meshwork-like structures at day 3 to 7. Moreover, calcium mineralization of clusters was observed, associated with the capacity for differentiation towards the osteoblastogenic or adipogenic lineage when cultured with differentiation media. Furthermore, the production of osteopontin (OPN) and angiopoietin-2 (Ang-2) was significantly higher in 3-D vs. 2-D MM-MSCs, assessed by multiplex luminex technology. Phenotypic profiling of 3-D MM-MSCs clusters revealed high expression of CD73+CD90+CD105+ and lack of expression of CD45, CD34 and HLA-DR, as in to 2-D MM-MSCs. MSC-specific markers including CD166 and HLA-ABC did not reveal any significant changes in 3-D vs. 2-D MM-MSCs; however, 3-D MM-MSCs had significantly decreased expression of CD271 and CD146 compared to 2-D cultures. We also observed significantly higher expression of extracellular matrix (ECM) molecules including fibronectin, laminin, collagen I, and collagen IV (p