Abstract Abstract 4026 Background: B cell maturation antigen (BCMA) is a receptor whose expression increases during B-cell development and is found on malignant cells from multiple myeloma (MM) patients; however, it has not been identified in human serum. Methods: Following informed consent (Western IRB BIO 001), serum was isolated from MM patients and analyzed with a BCMA enzyme-linked immunosorbent assay. Values represent the mean of triplicate experiments. Our human MM xenograft (LAGκ-2) was grown in SCID mice, and animals were treated with bortezomib (BORT) and cyclophosphamide (CY); tumor volume and BCMA levels were determined. Statistical significance of differences observed was determined using a Student's t test and analysis was determined using GraphPad prism software. Results: The serum BCMA levels from newly diagnosed MM patients (n = 58) was much higher (13.26 ng/ml) than among age-matched healthy subjects (n = 45; median 2.57 ng/ml; P < 0.0001) and MGUS subjects (n = 25; median 4.43 ng/ml; P = 0.002). Notably, protein levels were much higher among patients with relapsed or refractory disease (n = 88; median 18.99 ng/ml) compared to those with responsive (> partial response) disease (n = 95; median 3.48 ng/ml; P = 0.0016). Following treatment, patients with responsive disease showed decreases in BCMA levels whereas those with progressive disease showed increases. Additionally, with a median follow-up of 8 months (range, < 0 – 83 months), MM patients (n = 193) with BCMA levels above the median (8.43 ng/ml) showed a shortened survival compared to those with amounts below the median concentration (P < 0.0001). Following treatment with BORT and CY, we also showed a marked decrease in tumor volume and serum human BCMA levels in mice bearing the MM LAGκ-2 xenograft whereas untreated animals showed marked increases in tumor size and serum BCMA. Conclusions: This is the first report identifying serum BCMA in any human disease and suggests that these levels may be a novel biomarker for monitoring disease status and therapeutic response in MM patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 4986 Background: Janus kinase 2 (JAK2) is a cytoplasmic tyrosine kinase that carries out a series of cascading signals via signal transducer and activator of transcription (STAT)s, mitogen-activated protein kinase (MAPK), and phosphorylation of PI3K. Activation of the JAK2 pathway plays an important role in both normal and malignant hematopoiesis. The JAK pathway ha been shown to play a key role in multiple myeloma (MM). JAK2 has been specifically implicated in signaling by members of the type II cytokine receptor family (interferon [IFN] receptor), GM-CSF receptor (IL-3R, IL-5R, and GM-CSF-R), gp130 receptor family interleukin-6 (IL-6R) and single chain receptors (Epo-R, Tpo-R, GH-R, and PRL-R). IFN-α inhibits MM cell proliferation in association with cell cycle arrest at G1 and limits the clonogenic growth of both MM cell lines and primary MM patient specimens. SAR503 (Sanofi-Aventis) is a potent, highly selective JAK2 inhibitor. Thus, we evaluated the anti-MM effects of SAR503 as a single agent and in combination with other anti-MM drugs and evaluated gene and protein expression in MM cells exposed to these drugs. Experiment design: The MM cell lines RPMI8226, U266, and MM1s were cultured in RPMI1640 with standard nutrition supplements. Bone marrow aspirates were obtained from MM patients following informed consent. Bone marrow mononuclear cells (BMMCs) were isolated by using density-gradient centrifugation with Histopaque-1077 (Sigma, St Louis). Cells were plated in 96 well plates at a concentration of 6 × 10 4 cells/100 ml/well, and incubated for 24 hours prior to drug treatment, after which time the drugs were added in replicates of six for 48 hours. BMMCs were incubated in the presence of media, SAR503, doxorubicin, melphalan, dexamethasone, bortezomib, or IFN-α alone or the combination of SAR503 with one of these anti-MM agents. Following the 48-hour drug incubation, cell viability was assessed utilizing the cell proliferation MTS assay. For gene expression studies, total RNA was isolated MM tumor cells with or without drug exposure. RNA was reverse-transcribed into cDNA and amplified using the Thermo-Script RT-PCR System and PCR performed again using the GeneAmp PCR System 9700. Protein phosphorylation of MM tumor cells with or without drug exposure was determined with Western blot analysis. Results: SAR503 alone inhibited MM tumor cell proliferation in a concentration-dependent fashion. The 50% growth inhibition (IC 50 ) of cells from MM cell lines at 48 hours varied (IC 50 : RPMI8226 1mM; U266 0. 5mM; MM1s 10mM). IC 50 of primary MM tumor cells treated with SAR503 ranged from approximately 5 to 10mM in different patients. Notably, the combination of SAR503 and either doxorubicin or melphalan showed markedly reduced cell viability compared to either drug alone in all three MM cell lines and primary tumor cells from MM patients. Since this effect may have resulted from decreased cell proliferation due to inhibition of the JAK2 pathway and cell cycle arrest or increased cell death, we further determined cell apoptosis of MM tumor cells treated with SAR503 alone by using flow cytometric analysis to detect Annexin V and propidium iodide (PI) staining . Our data showed SAR503 increased MM tumor cell apoptosis in a concentration-dependent fashion. The combination of SAR503 and dexamethasone or bortezomib only slightly reduced tumor cell viability in both MM cell lines and primary MM tumor cells more than single agent treatment, and the combination of SAR503 with IFN-α did not enhance the anti-MM effects compared to single drug treatment. Notably, RT-PCR results showed marked decreases in both AKT1 and mTOR gene expression in MM tumor cells treated with SAR503. Conclusion: The combination of the JAK2 inhibitor SAR503 with doxorubicin or melphalan markedly reduces MM tumor cell viability more than single agent treatment. The results from these studies suggest that enhanced anti-MM activity may be observed when SAR503 is combined with conventional treatment for MM. We are currently evaluating the anti-MM effects of SAR503 in these combination treatments in vivo using our MM xenograft models. Disclosures: Berenson: Onyx: Consultancy, Honoraria, Speakers Bureau.

Abstract Abstract 1829 Background: Enhanced angiogenesis in multiple myeloma (MM) is an important step in its tumor growth, invasion, and metastasis into the bone marrow. The bone marrow is a complex and dynamic microenvironment with multiple cell types contributing to niches that support myeloma tumor cell proliferation. One of the important microenvironmental factors, hypoxia-inducible factor 1α (HIF-1α), has been known to be associated with increased angiogenesis and metastatic potential as well as poor prognosis in MM. However, the role of HIF-1α in the etiology, pathogenesis and possible treatment of MM remains unclear. LIM domain-containing protein (LIMD1), a tumor suppressor molecular links prolyl hydroxylases (PHD1, 2 or 3) to von Hippel–Lindau (VHL) as a molecular scaffold, PHDs-LIMD1-VHL. This results in down-regulation of HIF-1α. Experiment design: Following informed consent, ten bone marrow aspirates and five biopsies were obtained from MM patients and healthy subjects, respectively. Bone marrow mononuclear cells (BMMCs) were isolated by using density-gradient centrifugation with Histopaque-1077 (Sigma, St Louis). For gene expression studies, total RNA was isolated from BMMCs. RNA was reverse-transcribed into cDNA and amplified using the Thermo-Script RT-PCR System and PCR performed again using the GeneAmp PCR System 9700. Protein expression in BMMCs from MM and healthy subjects was determined using Western blot analysis and immunohistochemical (IHC) staining. Results: The results showed that LIMD1 gene expression in BMMCs as assessed using RT-PCR in MM patients was reduced compared to healthy subjects. In addition, PHD1 gene expression in BMMCs from MM patients was markedly reduced whereas PHD2 and PHD3 expression was similar to healthy subjects' BMMCs. The gene expression of VHL1, VHL2, and VHL3 in BMMCs was similar in MM patients compared to healthy subjects. Notably, LIMD1 gene expression in BMMCs from patients with monoclonal gammopathy of undetermined significance and primary amyloidosis patients was lover than among patients with progressive MM. Furthermore, the results of IHC staining showed LIMD1 protein expression in biopsy samples from MM patients with progressive disease was deceased and associated with high expression of HIF-1α. We also determined LIMD1 gene expression in MM patients treated with or without bortezomib. The results showed that this proteasome inhibitor did not affect LIMD1 expression. Conclusion and discussion: LIMD1 expression is reduced with down-regulation of PHD1 and up-regulation of HIF-1α in MM patients. The proteasome inhibitor did not inhibit LIMD1 gene expression in our experiments although it has reported that bortezomib inhibits HIF-1α levels in MM. The results from these studies suggest that LIMD1 may be an important key regulator of HIF1-1a and angiogenesis in MM. It has been reported that knocking out the LIMD1 gene will lead to over-expression of a single gene K-Ras and result in the development of lung adenocarcinoma. We are currently investigating the other pathways that are impacted by the reduction of LIMD1 expression, and how this may lead to the pathogenesis of MM. Disclosures: No relevant conflicts of interest to declare.

Preclinical and clinical studies have shown that proteasome inhibitors (PIs) have anti-MM activity in combination with dexamethasone or lenalidomide. However, no data exists on the anti-MM effects of combinations involving the PI delanzomib with dexamethasone and/or lenalidomide. Herein, we show that delanzomib in combination with dexamethasone and/or lenalidomide results in superior tumor reduction and extended tumor growth delays when compared to vehicle alone, these drugs alone, or the doublet of dexamethasone and lenalidomide. The favorable results obtained from the three xenograft studies suggest that delanzomib in combination with dexamethasone and lenalidomide should be explored for the treatment of MM.

SummaryAlthough TNFRSF17 (also designated as B‐cell maturation antigen (BCMA)) is expressed on tumour cells in B‐cell malignancies, it has not been found in serum. The present study found that BCMA concentrations were higher in the supernatants of cultured bone marrow mononuclear cells from multiple myeloma (MM) patients than in healthy subjects. Serum BCMA levels were measured in samples from MM patients (n = 209), monoclonal gammopathy of undetermined significance (MGUS) individuals (n = 23) and age‐matched controls (n = 40). BCMA was detected in the serum of untreated MM patients (n = 50) and levels were higher than in MGUS patients (P = 0·0157) and healthy subjects (P < 0·0001). Serum BCMA levels were higher among patients with progressive disease (n = 80) compared to those with responsive disease (n = 79; P = 0·0038). Among all MM patients, overall survival was shorter among patients whose serum BCMA levels were above the median (P = 0·001). We also demonstrated that sera from mice with human MM xenografts contained human BCMA, and levels correlated with the change in tumour volume in response to melphalan or cyclophosphamide with bortezomib. These results suggest that serum BCMA levels may be a new biomarker for monitoring disease status and overall survival of MM patients.

e18549 Background: B cell maturation antigen (BCMA) is a receptor whose expression increases during B-cell development and is found on malignant cells from multiple myeloma (MM) patients; however, it has not been identified in human serum. Methods: Bone marrow (BM) aspirates and peripheral blood were obtained from patients with MM, monoclonal gammopathy of undetermined significance (MGUS) and healthy control subjects following informed consent (Western IRB BIO 001). Serum was isolated and analyzed with a BCMA enzyme‑linked immunosorbent assay. Values represent the mean of triplicate experiments. BM mononuclear cells (MCs) were isolated using density‑gradient centrifugation and cultured for 72 h in RPMI1640 supplemented with 10% fetal bovine serum. Our human MM xenograft (LAGκ-2)was grown in SCID mice and treated with bortezomib (BORT) with cyclophosphamide (CY); tumor volume and BCMA levels were determined. Statistical significance of differences observed was determined using a Student’s t test and analysis was determined using GraphPad prism software. Results: We showed that the supernatants of cultured BMMCs from MM patients had high concentrations of BCMA (median = 2,250 pg/ml) whereas normal subjects showed minimal amounts (56 ng/ml; P < 0.0001). The serum BCMA levels from newly diagnosed MM patients (n = 51) had much higher levels (13.74 ng/ml) than among controls (n = 40; median 2.58 ng/ml; P < 0.0001) and MGUS subjects (n = 26; median 5.40 ng/ml; P = 0.005). Notably, protein levels were much higher among patients with relapsed or refractory (R/R) disease (n = 79; median 20.02 ng/ml) compared to those with responsive (> partial response) disease (n = 80; median 4.14 ng/ml; P = 0.0038). Following treatment, patients with responsive disease showed decreases in BCMA levels whereas those with R/R disease showed increases. Following treatment with BORT and CY, we also showed a marked decrease in tumor volume and serum human BCMA levels in mice bearing the LAGκ-2 xenograft whereas untreated animals showed marked increases in tumor size and serum BCMA. Conclusions: This is the first report identifying serum BCMA in any human disease and suggests that these levels may be a novel biomarker for monitoring disease status and therapeutic response of MM patients.

Purpose: Doxorubicin has shown efficacy especially in combination treatment for the treatment of multiple myeloma; however, its side effects limit its use. INNO-206 is an albumin-binding prodrug of doxorubicin, which is released from albumin under acidic conditions. Because INNO-206 has not been previously evaluated in any hematologic malignancy, we determined its anti–multiple myeloma effects. Experimental Design: The anti–multiple myeloma effect of INNO-206 at different pH levels on multiple myeloma cell proliferation using multiple myeloma cell lines with the MTS assay and antiangiogenic activity using the chorioallantoic membrane/feather bud assay were determined. The anti–multiple myeloma effects and toxicity of INNO-206 were also compared with conventional doxorubicin and PEGylated liposomal doxorubicin (PLD) alone, and in combination with bortezomib, using our multiple myeloma xenograft models. Results: INNO-206 inhibited blood vessel formation and reduced multiple myeloma cell growth in a pH-dependent fashion. INNO-206 alone produced marked anti–multiple myeloma effects in vivo at doses that doxorubicin was toxic, and the combination of INNO-206 plus bortezomib produced increased anti–multiple myeloma effects compared with either agent alone. In contrast, all mice receiving bortezomib with doxorubicin or PLD died. Conclusions: These findings show that INNO-206 produces anti–multiple myeloma effects in vitro and in vivo. It also enhances the antitumor effects of bortezomib. These results suggest that INNO-206 may provide patients with multiple myeloma with an anthracycline that may be administered safely at higher doses compared with free doxorubicin, resulting in superior efficacy compared with the currently available anthracyclines to treat this B-cell malignancy. Clin Cancer Res; 18(14); 3856–67. ©2012 AACR.

Abstract Lyn phosphorylation up-regulates several kinases including Syk, phospholipase Cγ2 and phosphatidyl inostitol-3 kinase through the immunoreceptor tyrosine-based activation motif (ITAM). Lyn plays critical roles in cell proliferation, Ca2+ mobilization and cell differentiation. This kinase also plays an essential role in transmission of inhibitory signals through phosphorylation of tyrosine residues within the immunoreceptor tyrosine-based inhibitory motifs (ITIM). ITAM-ITIM cross talk is important for macrophage differentiation and osteoclast formation. Bafetinib (INNO-406) is an inhibitor of Bcr-Abl, Fyn and Lyn kinase. No studies have evaluated potential anti-bone resorptive effects of this tyrosine kinase inhibitor. Osteoclasts are multinucleated bone-resorbing cells derived from monocytes that play critical roles in bone remodeling. To evaluate the effects of bafetinib on osteoclast formation and bone resorption, we isolated monocytes using immunomagnetic bead selection from normal or multiple myeloma (MM) patients' PBMCs. CD14+ cells (monocytes) were treated with 50ng/ml RANKL and 20 ng/ml MCSF at the beginning of the culture and during a medium change at 3 days. During the second day of culture, bafetinib or the nitrogen-containing bisphosphonate zoledronic acid, an inhibitor of osteoclast formation and bone resorption, was added into the cells. The cells were fixed and tartrate-resistant acid phosphatase staining performed on day 21. Bafetinib and zoledronic acid both markedly inhibited osteoclast cell formation of monocytes induced by RANKL and MCSF at similar concentrations in a concentration-dependent fashion in both monocytes derived from MM patients and normal subjects. Next, we assessed bone resorption using monocytes that were induced with M-CSF and RANKL and cultured on bone slides for 28 days. At that time, bone resorption was determined using toluidine blue staining. Resorption pits were measured and percentage of surface area with lacunar resorption on each bone slice was determined using an image analysis system. At a concentration as low as 5μM, bafetinib significantly inhibited bone resorption in a concentration-dependent fashion (P<0.001). We further examined the effects of bafetinib on NF-κB and JNK phosphorylation in human monocytes that were induced with RANKL and MCSF through assessment of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. Phospho-NF-κB protein levels were reduced and phosphorylation of JNKK was also decreased following exposure to bafetinib. These studies suggest that bafetinib may be a new therapeutic option to reduce skeletal complications through its ability to block osteoclast development and reduce bone resorption. We are evaluating the effects of this tyrosine kinase inhibitor on bone resorption in vivo using our SCID-hu murine models of human myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-305. doi:10.1158/1538-7445.AM2011-LB-305

Abstract Abstract 5102 We have recently reported that CGEN-928 is highly expressed on the cell membrane of cell lines, human xenografts, and primary tumor cells from MM. Anti-CGEN-928 (anti-TM21) polyclonal antibody blocked the expression of CGEN-928 which decreased MM tumor cell proliferation and increased apoptosis in the MM cell lines MM1s, RPMI8226 and U266 as well as primary MM tumor cells. The mechanism through which blocking CGEN-928 decreases MM tumor cell proliferation and enhances apoptosis has not been elucidated clear. In this study, a CGEN-928 shRNA (lentiviral particles) was used to silence this gene's expression, and determine its impact on the AKT signal transduction pathway which has been shown to play an important role in MM tumor cell metabolism proliferation, and survival. Briefly, MM1s or primary MM tumor cells were cultured in a 12-well plate for 24 hours prior to the viral infection. On the following day, a mixture of 5ug/ml Polybrene and fresh medium were added to the cells. The CGEN-928 shRNA lentiviral particles were then added to the culture. While transducing cells, we treated a portion of the cells with a negative control through introduction of control shRNA lentiviral particles. To ensure we achieved a successful transduction, we also treated another portion of the cells with cop GFP control Lentiviral particles. We confirmed that 75% of MM cells were transduced based on GFP+ cell counts after 24 hours treatment. The day following the transduction, the cultured medium was removed and replaced with fresh medium without polybrene. Two days following transduction, we used fresh 10ug/ml puromycin-containing medium to select stable MM cells. We replaced the medium with fresh puromycin-containing medium every three days until resistant MM tumor cells were stable. Proliferation rate of the MM1s tumor cells transduced with CGEN-928 shRNA (85%) 24 hours was much lower than the tumor cells transduced with control lentiviral particles rate (170%). The proportion of MM cells undergoing apoptosis treated with CGEN-928 shRNA (42%) was higher than MM cells transduced with control lentiviral particles (13%). We next examined several protein phosphorylation sites related to AKT signaling pathway by Western blot. The results showed AKT1 phosphorylation in MM tumor cells transduced with CGEN-928 shRNA or anti- CGEN-928 polyclonal antibody was decreased and phosphorylation of c-Raf, GSK-3β, factors downstream of AKT were also down-regulated. PTEN phosphorylation slightly decreased in MM cell treated with anti-CGEN-928 antibody but did not change in MM cells silenced with CGEN-928 shRNA. We further examined downstream gene expression of the AKT pathway when CGEN-928 was silenced using siRNA or the anti-CGEN-928 TM-21 antibody. We found AKT1 gene expression was reduced in the presence of CGEN-928 siRNA or antibody but it did not impact ATK2 and AKT3. mTOR gene expression in MM tumor cells was decreased with exposure to CGEN-928 siRNA but anti-TM21 showed no effect. Cyclin D1 gene expression in MM tumor cell was not affected by CGEN-928 siRNA and antibody. These studies suggest that blockage of CGEN-928 antigen expression inhibits MM tumor cell proliferation and enhance tumor cell apoptosis through AKT signaling pathway. Currently, a monoclonal anti-CGEN-928 antibody is in development that will be used by our group to evaluate its anti-MM effects both in vitro and in vivo using our SCID-hu models of human MM. Disclosures: Berenson: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Medtronic: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck: Research Funding; Genentech: Research Funding.

Abstract 2917 The inhibitory Fc receptor, Fc γRIIb, is expressed on plasma cells, controls their persistence in the bone marrow (BM) and their ability to produce serum Ig. Activation of Fc γRIIb leads to the phosphorylation of ITIM and recruitment of SH2-containing inositol 5'-phosphatase (SHIP) in plasma cells. Immunoreceptor tyrosine-based activation motif (ITAM) and ITIM provide the basis for two opposing signaling modules that duel for control of plasma cell activation. Fc γRIIb-mediated SHIP phosphorylation activates downstream ITAM or ITIM signaling. To determine whether multiple myeloma (MM) cells express Fc γRIIb, we performed immunohistochemical staining on bone marrow mononuclear cells from MM patients and controls. We found that not only CD20+ B cells expressed Fc γRIIb but more importantly CD138+ cells from MM patients also showed expression of this receptor. Next, we examined whether Fc γRIIb was present and expressed in CD138+ primary MM cells purified from fresh MM BM and the MM cell lines MM1s, RPMI8226, and U266 using PCR and RT-PCR on DNA and mRNA, respectively. We focused on the transmembrane domain of the Fc γRIIb gene with four primers from different parts of this domain since this portion plays a critical role in this receptor's function. The MM cell lines expressed different amounts of Fc γRIIb. Notably, we found that 17% (5/30) of MM patients showed absence of Fc γRIIb both using RT-PCR for mRNA and PCR for DNA. Moreover, use of these same primers on nonmalignant PBMCs from the MM patients also showed absence of this gene in the same five patients. As a result of these findings, we are currently sequencing Fc γRIIb in MM patients to determine if additional patients show mutational changes that affect the function of this receptor. We also further determined SHIP-1 phosphorylation using Western blot analysis since this protein mediates downstream signaling of Fc γRIIb. Following stimulation with Fc complexes, phosphorylation of SHIP-1 was markedly reduced in MM tumor cells compared to normal CD20+ B cells. Interestingly, the patients with missing Fc γRIIb expressed higher levels of SHIP-1 gene expression compared to patients with normal Fc γRIIb expression. We investigated the IgG-binding ability of MM patients (n=33) and normal donors (n=33) to Fc γRIIb. Each serum sample was incubated with cells from MHC1, a cell line that specifically expresses Fc γRIIb but not Fc γRI and Fc γRIIa. The results showed MM patients' serum IgG have much lower Fc γRIIb-binding ability than normal human IgG (P<0.05) by using both flow cytometric and immunofluorescence assays . Our findings suggest that the monoclonal protein produced by MM patients has a very low Fc γRIIb-binding ability and is incapable of signaling through the inhibitory ITIM pathway. Germline loss of Fc γRIIb in MM patients with variation in its expression level and its downstream signaling molecule SHIP and its phosphorylation as well as the inability of MM IgG to bind cells containing this receptor is a potential new mechanism that contributes to the uncontrolled growth of MM. Disclosures: Berenson: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Medtronic: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck: Research Funding; Genentech: Research Funding.

Abstract Abstract 2946 Introduction: We previously demonstrated the anti-MM effects of the proteasome inhibitor (PI) CEP-18770 with lenalidomide (LEN) and/or dexamethasone (DEX) using the MM xenograft model LAGκ-1B. Bortezomib has been shown to demonstrate synergistic anti-MM effects with both of these classes of drugs which has led to the successful use of these combinations to treat patients with MM. Thus, we conducted the current study to ascertain the anti-MM effects of CEP-18770 in combination with DEX and/or LEN in vivo using a different human severe combined immunodeficient (SCID) -hu MM model (LAGκ-1A) than then one previously evaluated and mentioned above (LAGκ-1B). Methods: Each naïve SCID mouse received a 20 – 40 mm3 MM tumor piece surgically implanted into the left hind limb superficial gluteal muscle. Seven days post-implantation mice were randomized into treatment groups based on human immunoglobulin G (IgG) levels. CEP-18770 (4 mg; Cephalon, Inc., Frazer, PA, USA) stock solution was dissolved in propylene glycol (800 μl) and added to 5% mannitol to generate a final stock solution of 1 mg/ml, diluted (5% mannitol). Mice were injected with 1 mg/kg twice weekly (T, Th) via intravenous (i.v.) injection. LEN stock solutions were prepared daily from pills and diluted in 5% carboxymethylcellulose. DEX was obtained as a 4 mg/ml stock solution, diluted (0.9% sodium chloride). Mice were treated daily with DEX at 1.25 mg/kg via intraperitoneal (i.p.) injection and 30 mg/kg of LEN via oral gavage. On a weekly basis, tumor size was measured using standard calipers (n = 9–10 mice/group) and human IgG levels with an ELISA (Bethyl Laboratories, Montgomery, TX). This study was conducted according to protocols approved by the Institutional Animal Care and Use Committee. Results: At day 35 post tumor implantation, mice receiving the doublets of CEP-18770 plus daily dexamethasone or lenalidomide and the triplicate regimen containing all three drugs markedly inhibited tumor volume growth compared to mice receiving vehicle. Notably, P -values could not be calculated because all mice receiving the doublet and triplicate combination regimens had undetectable tumor volumes (zero tumor volume measurements) and thus a t-test could not be calculated. These tumors remained undetectable beginning at day 35 and throughout the study until its termination (day 91). CEP-18770 or DEX administered alone also significantly inhibited tumor volume growth (P = 0.0005, P = 0.0205, respectively) at this same time point, when compared to mice receiving vehicle, whereas LEN alone did not. However, in contrast to the tumors which eventually grew in mice after single agent treatment with CEP-18770, DEX, or LEN and the doublet LEN + DEX group, tumors did not reappear among mice which received the CEP-18770 doublet or CEP-18770 triplicate combination regimens. Similar inhibitory effects were obtained for IgG levels as those observed for tumor volume growth inhibition. Mice receiving CEP-18770 doublets (CEP-18770 + DEX or LEN) or all three drugs together were sacrificed at day 91. Overall, 10/10 mice survived the CEP-18770 + DEX regimen, and 9/10 mice survived in both the CEP-18770 + LEN and CEP-18770 + DEX + LEN groups. Conclusions: These in vivo studies using our LAGk-1A SCID-hu model show that CEP-18770 administered alone, in combination with DEX or LEN, or in triplicate combination with both DEX and LEN, resulted in significant inhibition of tumor growth as determined by measuring tumor volume and IgG levels when compared to vehicle-treated and single agent treated mice. Although the initial anti-MM effects were similar between single agent CEP-18770 and the doublet and triplicate combination therapies, with longer follow-up the doublet and triplicate combination therapies proved to be superior. The toxicity profile was negligible and similar between CEP-18770 monotherapy, combined with either DEX or LEN, and the triplicate combination regimen. Pre and post-treatment body weight comparisons of these groups demonstrated that mice receiving the different treatments gained weight to a similar extent during study treatment. This study demonstrates that using a different MM model (LAGκ-1A), the PI CEP-18770 in combination with DEX and/or LEN results in significant tumor growth and IgG inhibition, and provides further support for the development of the novel agent for the treatment of MM. Disclosures: Berenson: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Medtronic: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck: Research Funding; Genentech: Research Funding.

Abstract Abstract 455 Functional B cell and plasma cell immune responses are dependent upon an exquisitely controlled process integrating signals from activating and inhibitory receptors present on the surface of these cells. These activating and inhibitory signaling pathways regulate both the quality and quantity of immunoglobulin (Ig) production. These signaling motifs, termed immunoreceptor tyrosine-based activation motif (ITAM) and ITIM provide the basis for two opposed signaling modules that duel for control of plasma cellular activation within the immune system. The inhibitory Fc receptor FcγRIIb is expressed on plasma cells and controls their persistence in the bone marrow and their ability to produce most serum Ig. Activation of FcγRIIb leads to the phosphorylation of ITIM and recruitment of SH2-containing protein tyrosine phosphatase-1 (SHP-1) SHP-2 and the SH2-containing inositol 5-phosphatase (SHIP) in plasma cells. FcγRIIb-mediated SHIP phosphorylation results in enhanced degradation of PtdIns (3,4,5)P, which is required for B-cell antigen receptor (BCR)-induced recruitment and activation of downstream ITAM signaling molecules. The inhibitory IgG Fc receptor FcγRIIB was the first discovered and remains the best studied example of an ITIM-containing receptor. In this study, we first investigated the IgG-binding ability of 18 MM patients and 10 normal donors to FcγRIIb using flow cytometric analysis. Each serum sample was incubated with MHC1 cells that only express FcγRIIb but do not express FcγRI and FcγRIIa. After washing three times with 1 × PBS, anti-human IgG antibody conjugated with FITC was added to the cells for another 30 minutes. The results showed MM patients' serum IgG have much lower FcγRIIb-binding ability than normal human IgG (P<0.05). Immunohistochemical staining also demonstrated MM patients' sera showed obviously less binding to the FcγRIIb on MHC1 cells compared to serum samples from normal human donors. We further analyzed the FcγRIIb-SHIP signaling pathway in normal B-cells following exposure to MM and normal human sera. Fresh human B-cells were isolated using anti-CD20 antibodies with magnetic bead selection and the cells were washed in an acidic solution (pH 4) for 1 minute to remove any bound antigen from the cell surface. Raji B-cells were also acid-treated. First, these cells were exposed to MM patients' or normal human sera for 5, 15, 30, or 60 minutes to determine the maximum time point of SHIP phosphorylation. Using a standard Western blot protocol, phosphorylated SHIP and total SHIP protein expression were visualized using an enhanced chemiluminescence detection system. The maximum time point of SHIP phosphorylation was 15 minutes. The results showed that both normal human B-cells and Raji B-cells showed markedly lower SHIP phosphorylation/total SHIP following exposure to MM patient serum compared to normal human serum which is consistent with the lack of binding of human Ig to FcγRIIb. Our findings suggest that the monoclonal protein produced by MM patients has very low FcγRIIb-binding ability and is incapable of signaling through inhibitory ITIM pathway. Most importantly, FcγRIIb is expressed on plasma cells and controls their persistence in the bone marrow in addition to Ig production. Cross-linking of FcγRIIb induces apoptosis of plasma cells (Xiang Z et al Nat Immunol 8: 419-29, 2007). Thus, it is possible that this effect prevents the induction of apoptosis in MM cells. Previous studies have reported that normal Ig is capable of inducing B-cell apoptosis. Thus, it is possible that exposure of MM cells to normal immunoglobulin may be capable of inducing apoptosis of these cells and reducing their production of M-protein. We are currently evaluating the ITIM signal transduction pathway, M-protein production and apoptosis in MM and normal B-cells following exposure to normal and MM M-protein IgG. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 5015 Lyn is a member of the Src kinase (SFK) family and controls the activation threshold of multiple signaling pathways in different types of cells, including B cells (Yamanashi et al., 1989, PNAS). Multiple myeloma (MM) is a hematological malignancy of plasma cells. In MM, Lyn is involved in IL-6-induced cell proliferation (Hallek et al., 1997, Exp Hematol). As one of the most important growth and anti-apoptotic factors for MM cells, both the IL-6/STAT3 and IL-6/ERK pathways have been shown to promote MM cell proliferation and prevent apoptosis. However, STAT3 and ERK activation is not sufficient for IL-6-induced proliferation, which further requires the activation of SFK (Ishikawa et al., 2002, Blood). IL-6-induced proliferation of U266 myeloma cells has been reported to be significantly suppressed following exposure to Lyn-specific antisense oligonucleotides or a Src kinase inhibitor (Ishikawa et al., 2002, Blood). Therefore, a Lyn specific inhibitor, either alone or in combination with other anti-MM drugs, may be a more effective regimen for the treatment of MM. INNO-406 is a novel inhibitor of ABL as well as Lyn. In laboratory-based studies, it has shown anti-tumor activity in hematological malignancies, especially in leukemia cells harboring BCR-ABL mutations (Kimura et al., 2005, Blood). Since Lyn is involved in MM cell proliferation, targeting Lyn may be an alternative therapeutic approach and improve the efficacy of other anti-MM agents. Thus, we conducted this study in order to determine the anti-MM effects of INNO-406. First, Western blot analyses demonstrated that Lyn was expressed in all MM cells analyzed, including bone marrow (BM) mononuclear cells derived from MM patients and MM cells from LAGκ1A, LAGκ1B and LAGκ2 xenografts, three of our unique in vivo SCID-hu models of human MM, which were originally developed from MM patient BM biopsies and have been maintained in SCID mice. However, Lyn activation, as demonstrated by phosphorylation of its downstream molecule HS1, was infrequently found in MM cells. INNO-406 inhibited the growth of the three MM cell lines tested including RPMI8226, U266 and MM1S. Interestingly, the U266 cell line is most resistant to INNO-406 among the three MM cell lines tested although its Lyn activation and expression level is the highest. Apoptosis analysis using flow cytometric analysis following Annexin V staining demonstrated that INNO-406 induced apoptosis in MM cells. When combined with melphalan, doxorubicin or bortezomib, INNO-406 synergistically inhibited MM cell growth. We are currently studying the molecular mechanisms of INNO-406's anti-MM effect, either alone or in combination treatment, and validating our in vitro findings using our unique in vivo mouse models of human MM. Updated data from these additional studies will be reported at the meeting. Disclosures: No relevant conflicts of interest to declare.

Enhanced angiogenesis is a hallmark of solid tumors and hematological malignancies. Anti-angiogenic therapeutic approaches have recently been shown to be effective for the treatment of certain cancers. Endothelial cells migrating to tumors provide them with new blood vessels that are critical for their growth and survival. We have developed a novel and rapid method to evaluate the anti-angiogenic activity of new agents consisting of a combined chorioallantoic membrane (CAM) and feather bud (FB) assay. Unlike previous assays, this new assay assesses the effects of drugs on the ability of tissues to attract and develop their own blood supply. The CAM already has a well-developed vascular network that is capable of providing blood vessels to the non-vascularized FB, allowing for this tissue to develop feathers. As a result, the exposure of the FB to drugs for 2 days followed by attachment to the CAM for 4 days allows evaluation of the compound's ability to impact blood vessel and feather formation within the CAM-attached FB tissue. Feather formation is determined as well as expression of endothelial cell genes and proteins analyzed. Using agents with known anti-angiogenic activity including fumagillin, minocycline, zoledronic acid, doxorubicin and agents lacking anti-angiogenic activity such as melphalan, we have shown that the CAM/FB assay can accurately and rapidly assess the ability of agents to prevent blood vessel and feather development within non-vascularized tissues.

Cancer stem cells persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Development of specific therapies targeted at cancer stem cells gives hope for improvement in the survival and quality life of cancer patients. Multiple myeloma (MM) is a cancer characterized by clonal expansion of terminally differentiated B cells. In order to characterize whether cancer stem cells can be identified in these patients, fresh bone marrow biopsies with 90% MM cells from MM patients were implanted into the superficial gluteal muscle of C.B-17 severe combined immunodeficient (SCID) mice. The tumors were excised from donor mice two months following implantation, and digested with proteinase-E to produce a single cell suspension. These cells were analyzed using flow cytometry to identify specific cellular phenotypes within the tumor population. Approximately 13% of the tumor cells were CD138+ cells, 1–2% CD20+ cells and 2–3% CD133+ cells. To examine gene expression within these populations, we isolated the tumor cells using immunomagnetic bead selection. Cells (1X108) were incubated with 200ml of anti-CD138 microbeads and either anti-CD133 or CD20 microbeads. The cell suspension was applied to the magnetic column and unbound cells were passed through the column by washing followed by centrifugation, and finally resuspended. Total RNA was purified from the cells and gene expression of each population was examined using RT-PCR analysis of specific previously identified stem cell-related transcription factors. β-catenin plays a critical role in stem cell development; and, furthermore, the Wnt-β-catenin signaling pathway is important for maintaining the balance of proliferation versus differentiation in the stem cell population. The gene expression of KLT-4, Oct-4, SOX2, and C-myc has recently been shown to convert nonterminally differentiated B cells into a pluripotent stem cell state. In our studies, we found that the CD20+/CD138− and CD133+/CD138− subpopulations both expressed high levels of β-catenin, KLT-4, Oct-4, SOX2, and C-myc. These small populations of tumor cells are likely to represent MM cancer stem cells as they express genes consistently identified in cancer stem cells identified in other types of cancers. We unexpectedly found that CD138+ cells also expressed β-catenin, KLT-4, Oct-4, SOX2, and C-myc. This population of cells might be a “premature” tumor cell in MM at a middle stage of tumor cell differentiation which ultimately differentiates into a mature MM cell. Only CD20−/CD138− cells showed no expression of β-catenin, KLT-4 and SOX2 and markedly reduced Oct-4 gene expression whereas the amount of C-myc gene expression was similar to the levels in the other tumor cell subtypes. Only CD133−/CD138− cells lost β-catenin and showed a reduction in Oct-4 gene expression but still expressed the KLT-4, SOX2, and C-myc genes. To further examine these cancer stem cell and mature tumor cell populations in terms of growth in vivo, we have injected subcutaneously CD20+/CD138−, CD133+/CD138−, CD20−/CD138−, and CD133−/CD138− tumor cell subpopulations back into SCID mice. We will assess growth of cells from these subtypes in vivo as determined by changes in tumor volume and Ig protein levels. We also will determine the sensitivity of these subtypes in vivo to treatment with a variety of agents with anti-MM activity including bortezomib, lenalidomide, melphalan, and Doxil. These studies have uncovered specific subpopulations within the tumor clone of MM and identified differences in expression of genes known to be involved in stem cell function. Further work should lead to specific treatments that can effectively treat these different subpopulations within the tumor clone in MM.

Abstract 3050 Poster Board II-1026 We have previously shown that tumor cells from multiple myeloma (MM) patients express pleiotrophin (PTN). This protein is elevated in the serum of MM patients and we have shown that, in combination with M-CSF, it induces the transdifferentiation of monocytes into endothelial cells both in vitro and in vivo (Chen et al. Blood 2009). In this study, we determined the level of PTN expression in a variety of solid tumor types including breast, ovarian, prostate, and pancreatic cancers, and examined whether PTN produced by tumor cells from these different types of solid tumors could also induce transdifferentiation of human monocytes into endothelial cells both in vitro and in vivo . Our results showed that breast, ovarian, prostate, and pancreatic cancers all produce high levels of PTN as determined by RT-PCR, Western blot analysis, immunohistochemical staining, and measurement of the level of this growth factor secreted into the medium derived from cultured tumor cells whereas normal peripheral blood mononuclear cells (PBMCs) showed no expression of this protein. Next, monocytes derived from CD14-selected PBMCs or the THP-1 cell line were exposed to breast, ovarian, prostate, and pancreatic cancer cells on collagen I-coated Transwell plates with M-CSF. Following culture of these cells for 5-14 days, both fresh CD14+ cells and THP-1 cells changed their morphology into endothelial-like cells and expressed the endothelial genes and proteins Flk-1 and Tie-2 as determined with RT-PCR and Western blot analysis, respectively. These endothelial cell-inducing effects on monocytes were blocked with anti-PTN antibodies. Next, we determined whether human monocytes could be incorporated into blood vessels and express endothelial cell markers in vivo within solid tumors that express PTN. Human breast cancer cells (MDA-MB-231) alone, THP-1 monocytes transduced with the green fluorescent protein (GFP) gene, or the combination of both cell types were injected subcutaneously into severe combined immunodeficient (SCID) mice. Mice were sacrificed 8 weeks later and the tumor tissue was fixed and cut into frozen sections. Breast cancer cells or GFP+ THP-1 monocytes alone did not demonstrate the presence of GFP-marked cells within tumor blood vessels. When breast cancer cells and GFP+ THP-1 cells were injected together, GFP-marked cells were found within tumor blood vessels; and, moreover, double staining of serial sections of the breast cancer with anti-Tie-2 and CD31 antibodies showed a similar distribution pattern of staining as the blood vessel cells showing the presence of GFP. We also examined endothelial gene expression in these samples using RT-PCR. The results showed that the THP-1 monocytes alone or breast cancer cells alone did not express endothelial genes whereas THP-1 monocytes mixed with breast cancer cells showed endothelial gene (FLK-1, Tie-2) expression. These data show that solid tumors through expression of PTN support new blood vessel formation by the transdifferentiation of monocytes into endothelial cells and provide a new potential target for inhibiting early blood vessel formation within tumors. Disclosures No relevant conflicts of interest to declare.

Enhanced angiogenesis is a hallmark of cancer. Pleiotrophin (PTN) is an angiogenic factor that is produced by many different human cancers and stimulates tumor blood vessel formation when it is expressed in malignant cancer cells. Recent studies show that monocytes may give rise to vascular endothelium. In these studies, we show that PTN combined with macrophage colony-stimulating factor (M-CSF) induces expression of vascular endothelial cell (VEC) genes and proteins in human monocyte cell lines and monocytes from human peripheral blood (PB). Monocytes induce VEC gene expression and develop tube-like structures when they are exposed to serum or cultured with bone marrow (BM) from patients with multiple myeloma (MM) that express PTN, effects specifically blocked with antiPTN antibodies. When coinjected with human MM cells into severe combined immunodeficient (SCID) mice, green fluorescent protein (GFP)-marked human monocytes were found incorporated into tumor blood vessels and expressed human VEC protein markers and genes that were blocked by anti-PTN antibody. Our results suggest that vasculogenesis in human MM may develop from tumoral production of PTN, which orchestrates the transdifferentiation of monocytes into VECs.

Abstract 611 Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating NF-κB and JNK signal transduction pathway resulting in inhibition of tumor cell proliferation and osteoclast formation. The unique biological function of TRAF6 is largely determined within its TRAF-C domain which does not interact with peptide motifs that are recognized by other TRAFs including 1, 2, 3 or 5. We have recently reported inhibition of cell proliferation and increased apoptosis of multiple myeloma (MM) cells through regulation of the NF-κB and JNK pathways through silencing the TRAF6 C-domain mRNA. In this study, we determined the effects of TRAF6 dominant negative peptides on MM cells, osteoclast formation and bone resorption. We cloned a 167 amino acid (in residues 333 to 508) fragment to produce a TRAF6 negative dominant (TRAF6dn) construct and synthesized an inhibitory decoy peptide of the TRAF6 interaction domain with CD40 and another peptide interacting with the TRAF6-RANK binding domain as well as a control peptide. All peptides were synthesized with a 16 amino acid permeable peptide. Using the MM1s, RPMI8226, and U266, we evaluated the effects of these peptides on MM tumor cell growth using an MTS assay and apoptosis with an Annexin V assay. We found that TRAF6dn peptides significantly inhibited MM cell proliferation maximally at 72 hours whereas effects on induction of apoptosis in MM cells were most prominent at 48 hours. The decrease in cell proliferation and increase in cell apoptosis occurred in a concentration-dependent fashion. We found that TRAF6dn also markedly inhibited osteoclast cell formation from freshly derived human monocytes induced by RANKL and M-CSF in a concentration-dependent fashion comparing with cells exposed to control peptide. We further examined the effects on MM cell apoptosis of the TRAF6 decoy or CD40 decoy peptides alone and in cells exposed to the combination of both peptides. The results showed either decoy peptide alone slightly induced apoptosis of MM tumor cells whereas the combination of both peptides demonstrated marked apoptosis of MM cells. We also showed that although melphalan alone induced apoptotic cell death, this effect was markedly enhanced when this alkylating agent was combined with the TRAF6 decoy peptide. Although the CD40 peptide alone did not inhibit osteoclast formation, TRAF6 decoy peptide alone and the combination of both decoy peptides markedly inhibited formation of these bone resorbing cells. We also examined the effects of TRAF6dn on NF-κB and JNK by measuring JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. Phospho-NF-κB protein levels and phosphorylation of JNKK are both markedly reduced when MM cells are exposed to TRAF6dn fragment or TRAF6 decoy peptide. These studies suggest that TRAF6dn or the combination of TRAF6 decoy and CD40 decoy peptides may be excellent targets to block both myeloma cell and osteoclast cell formation. The study has been extended to assess the effects of these peptides in vivo using our SCID-hu murine model of human myeloma. Disclosures: No relevant conflicts of interest to declare.

Enhanced angiogenesis is a hallmark of solid tumors and hematological malignancies, and anti-angiogenic therapeutic approaches have recently shown significant benefit in the clinic. As a result, many anti-angiogenic agents are currently in early development. Very few methods have been used to evaluate the anti-angiogenic activity of these agents using an ex vivo assay. Unfortunately, currently available methods are both time consuming and costly. We have developed a novel approach to test the anti-angiogenic activity of new agents in a rapid, accurate and inexpensive way. This model consists of using a combined chorioallantoic membrane (CAM) and feather bud (FB) assay. The CAM already has a well developed vascular network and provides an ideal microenvironment and the FB serves as an active biological testing tool for evaluating angiogenesis. FB is a component of epithelial and mesenchymal cells. The method consists of using fertilized chick eggs incubated horizontally at 37.5°C in a humidified incubator and windowed by day 8. Another set of E8 chicken embryonic skins are collected under a dissecting microscope to isolate FB. The FB is treated with drugs or control reagents and implanted onto the CAM. The eggs are sealed with an adhesive tape and incubated for an additional 2–4 days. The endothelial cells of CAM proliferate and migrate into the FB after two days. After 4 days of culture, both blood vessel formation and FB development are determined by microscopy. New blood vessels in FB are analyzed by H&E and immunohistochemical (IHC) staining and expression of endothelial genes and proteins using RT-PCR and Western blot analysis, respectively. First, we establish that the compound being tested should only affect endothelial proliferation or migration and not kill the epithelial and mesenchymal tissues. We have used this new method to investigate several compounds. First, we evaluated the anti-angiogenic agent fumagillin (1μM) and minocycline (100nM). Although neither drug had any cytotoxic effects on the epithelial and mesenchymal tissues when cultured alone, marked inhibition of FB development occurred on the CAM in a dose-dependent fashion with both drugs as determined by microscopy and IHC. In addition, Western blot analysis showed marked inhibition of Tie-2 protein expression in a dose-dependent fashion in the presence of these drugs. Zoledronic acid, a potent bisphosphonate which has recently been shown to harbor anti-angiogenic activity, was found to markedly inhibit FB development in the presence of this drug at a concentration of 10 μM whereas less effect was observed at 2 μM. This drug did not have any direct effect on epithelial and mesenchymal cells when these tissues were cultured alone. We then examined gene and protein expression of the FB cells on CAM that were treated with zoledronic acid. Both FLK-1 and Tie-2 transcript and protein levels were significantly reduced in a dose-dependent fashion following treatment with zoledronic acid as assessed by RT-PCR and Western blot analysis. We are currently testing the potential anti-angiogenic effects of many other novel drugs using this new model. Overall, the present findings demonstrate that the CAM/ FB angiogenesis model is likely to be a reliable, fast, sensitive, and economical system to screen the anti-angiogenic effects of new agents.