Murine Model Of Multiple Myeloma Research Articles

Single-Dose Anti-CD138 Radioimmunotherapy: Bismuth-213 is More Efficient than Lutetium-177 for Treatment of Multiple Myeloma in a Preclinical Model.

Radioimmunotherapy (RIT) has emerged as a potential treatment option for multiple myeloma (MM). In humans, a dosimetry study recently showed the relevance of RIT using an antibody targeting the CD138 antigen. The therapeutic efficacy of RIT using an anti-CD138 antibody coupled to (213)Bi, an α-emitter, was also demonstrated in a preclinical MM model. Since then, RIT with β-emitters has shown efficacy in treating hematologic cancer. In this paper, we investigate the therapeutic efficacy of RIT in the 5T33 murine MM model using a new anti-CD138 monoclonal antibody labeled either with (213)Bi for α-RIT or (177)Lu for β-RIT. A new monoclonal anti-CD138 antibody, 9E7.4, was generated by immunizing a rat with a murine CD138-derived peptide. Antibody specificity was validated by flow cytometry, biodistribution, and α-RIT studies. Then, a β-RIT dose-escalation assay with the (177)Lu-radiolabeled 9E7.4 mAb was performed in KalwRij C57/BL6 mice 10 days after i.v. engraftment with 5T33 MM cells. Animal survival and toxicological parameters were assessed to define the optimal activity. α-RIT performed with 3.7 MBq of (213)Bi-labeled 9E7.4 anti-CD138 mAb increased median survival to 80 days compared to 37 days for the untreated control and effected cure in 45% of animals. β-RIT performed with 18.5 MBq of (177)Lu-labeled 9E7.4 mAb was well tolerated and significantly increased mouse survival (54 vs. 37 days in the control group); however, no mice were cured with this treatment. This study revealed the advantages of α-RIT in the treatment of MM in a preclinical model where β-RIT shows almost no efficacy.

Surgical Control/Cure of 5TGM1 Murine Multiple Myeloma Model by Thyroparathyroidectomy

The 5TGM1 transplanted C57BL6/KaLwRij mouse is widely used to study murine multiple myeloma development. It recapitulates many features of human multiple myeloma including monoclonal paraprotein as well as bone lesions. Using this mouse model we demonstrated the crucial role of the parathyroid hormone 1 receptor (PTH1R) on the survival of mice exposed to proteasome inhibitor drugs such as bortezomib and carfilzomib; specifically the blockage of the PTH1R function by parathyroid hormone antagonist (PTH 7-34) resulted in significant abrogation of the beneficial survival effect of proteasome inhibitor drugs. To further investigate the effect of the parathyroid hormone (PTH) axis on murine myeloma development we performed thyroparathyroidectomy on the C57BL6/KaLwRij mouse before or after 5TGM1 transplantation. Thyroparathyroidectomy was performed by trained personnel in general anesthesia with isoflurane; all surgical/postsurgical procedures and animal welfare followed an IACUC approved protocol at the University of Arkansas. To prevent hypocalcaemia at postsurgery, 1M CaCl2 solution in the drinking bottle was supplemented for a week. All tested mice received 0.5X106 5TGM1 cell infusion by intravenous injection; mice were divided into three groups; Group 1 control mice (n=10) received 5TGM1 infusion at day 0; Group 2 included 15 mice which on day 10 post 5TGM1 cell infusion underwent thyroparathyroidectomy; Group 3 included 15 mice which at 20 days post-surgery received infusion of 5TGM1 cells. 30% of animals experienced post-surgical complications and died in the first 7 days from procedure; those animals were not included in this survival analysis which is based on 10 animals for each cohort. Blood samples were collected at weekly intervals for IgG2 measurements. The survival curves of mice recovered after surgery are shown in the figure. All control mice (group 1) developed myeloma progression and died within 3 weeks post-transplant; 2 mice from group 2 and 5 mice of the third cohort manifested disease. All 7 animal deaths were pathologically and serologically attributed to myeloma progression. Compared to controls thyroparathyroidectomized mice (Group 2 and Group 3) showed significantly longer survival p Disclosures Zangari:Norvartis: Membership on an entity9s Board of Directors or advisory committees; Onyx: Research Funding; Millennium: Research Funding.

Abstract 786: Effects of combination treatment with cabozantinib and bortezomib in the 5TGM1 murine multiple myeloma model

Abstract Cabozantinib (cabo) is an inhibitor of tyrosine kinases including MET, VEGFR2, RET, and the TAM family kinases TYRO3, AXL, and MER, and has shown clinical activity in patients with castration-resistant prostate cancer and other solid tumors with bone metastases. Multiple myeloma (MM) is a monoclonal B-cell (plasma cell) neoplasia representing ∼2% of all cancer deaths. The clinical hallmark is presence of multiple osteolytic lesions causing bone pain, pathologic fractures, and hypercalcemia. Circulating levels of HGF and VEGF are upregulated in MM patients, and regulation of plasma cell-osteoblast communication by the HGF-MET signaling pathway has been implicated in the development of lytic bone disease in these patients. We have previously shown that cabo is active in the syngeneic 5TGM1 mouse MM model. This study aimed to determine whether combination with bortezomib would yield additional benefit. Four experimental groups were included: 1) Control group receiving vehicle, 2) Bortezomib (0.5 mg/kg ip twice a week), 3) Cabo (10 mg/kg, PO QD) and 4) Combination (bortezomib + cabo, same doses). Female C57BL/KaLwRij mice were allocated to treatment groups (n = 15 per group) with equivalent average body weights. At day 0, animals were inoculated with 5TGM1 cells by IV administration. Dosing began at day 1 and continued daily until euthanasia. Body weights were determined twice a week and blood samples were collected at days -1, 16, 23, 35 and at sacrifice for analysis of paraprotein (IgG2b), PINP and TRACP 5b. Development of osteolytic lesions was detected by radiography at day 35 and at sacrifice. Mice were sacrificed individually when they became paraplegic, lost over 20% of body weight, or had severe breathing problems. The maximum length of the study was 70 days. By study day 35, the osteolytic lesions were not affected by bortezomib, were reduced by cabo alone, and further reduced by the combination treatment. Bortezomib had inhibited the rise in serum IgG2b levels, but cabo and the combination treatment had not. Despite the effects on serum IgG2b, bortezomib did not significantly increase survival, whereas cabo and the combination treatment did. Increased survival with the combination was significant when compared to bortezomib monotherapy, but not when compared to cabo monotherapy. Earlier we have shown that cabo dose-dependently increases the necrotic tumor area in bone, and proposed that the rise in IgG2b was due to lysis of plasma cells and not tumor growth. Consistent with this hypothesis, the IgG2b levels of cabo treated mice were lower at sacrifice than at day 35 in this study. In summary, cabo increased survival and exhibited bone-protective and anti-tumor effects in this murine model of MM. Combination with bortezomib showed additive effects on survival. Based on these results, further investigation of cabozantinib in multiple myeloma is warranted. Citation Format: Mari I. Suominen, Katja M. Fagerlund, Esa Alhoniemi, Jukka P. Rissanen, Jussi M. Halleen, Dana T. Aftab. Effects of combination treatment with cabozantinib and bortezomib in the 5TGM1 murine multiple myeloma model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 786. doi:10.1158/1538-7445.AM2015-786

Therapeutic targeting of N‐cadherin is an effective treatment for multiple myeloma

Elevated expression of the cell adhesion molecule N-cadherin (cadherin 2, type 1, N-cadherin (neuronal); CDH2) is associated with poor prognosis in newly-diagnosed multiple myeloma (MM) patients. In this study, we investigated whether targeting of N-cadherin represents a potential treatment for the ~50% of MM patients with elevated N-cadherin. Initially, we stably knocked-down N-cadherin in the mouse MM plasma cell (PC) line 5TGM1 to assess the functional role of N-cadherin in MM pathogenesis. When compared with 5TGM1-scramble-shRNA cells, 5TGM1-Cdh2-shRNA cells had significantly reduced adhesion to bone marrow endothelial cells. However, N-cadherin knock-down did not affect 5TGM1 cell proliferation or adhesion to bone marrow stromal cells. In the C57BL/KaLwRij murine MM model, mice intravenously inoculated with 5TGM1-Cdh2-shRNA cells showed significantly decreased tumour burden after 4 weeks, compared with animals bearing 5TGM1-scramble-shRNA cells. Finally, the N-cadherin antagonist ADH-1 had no effect on tumour burden in the established disease setting, whereas up-front ADH-1 treatment resulted in significantly reduced tumour burden after 4 weeks. Our findings demonstrate that N-cadherin may play a key role in the extravasation of circulating MM PCs promoting bone marrow homing. Moreover, these studies suggest that N-cadherin may represent a viable therapeutic target to prevent the dissemination of MM PCs and delay MM disease progression.

Impact of the immunomodulating peptide thymosin alpha 1 on multiple myeloma and immune recovery after hematopoietic stem cell transplantation.

Multiple myeloma (MM) is characterized by the accumulation of monoclonal plasma cells in the bone marrow and causes several immune alterations in patients. Thymosin α1 (Tα1) is a thymic peptide that has been associated with immuno-stimulating properties. In addition, this peptide exerts anti-tumor effects in several cancer types. Beneficial effects of Tα1 administration have also been shown on immune reconstitution after hematopoietic stem cell transplantation (HSCT), a current treatment modality in hematological malignancies including MM. In this study, we observed a slight reduction in the proliferation of murine and human MM cell lines in the presence of Tα1 in vitro. However, using two immunocompetent murine MM models (5TGM1 and MOPC315.BM), we did not observe any impact of Tα1 administration on MM development in vivo. Furthermore, no beneficial effects of Tα1 treatment were observed on lymphocyte immune reconstitution after transfusion of human hematopoietic stem cells into immunodeficient mice. In conclusion, despite direct effects of Tα1 on human MM cell line proliferation in vitro, Tα1 did not exert anti-myeloma effects in vivo in the two murine models tested. Moreover, Tα1 failed to improve immune recovery in a xenogeneic HSCT model.

Glycosphingolipid synthesis inhibition limits osteoclast activation and myeloma bone disease.

Glycosphingolipids (GSLs) are essential constituents of cell membranes and lipid rafts and can modulate signal transduction events. The contribution of GSLs in osteoclast (OC) activation and osteolytic bone diseases in malignancies such as the plasma cell dyscrasia multiple myeloma (MM) is not known. Here, we tested the hypothesis that pathological activation of OCs in MM requires de novo GSL synthesis and is further enhanced by myeloma cell-derived GSLs. Glucosylceramide synthase (GCS) inhibitors, including the clinically approved agent N-butyl-deoxynojirimycin (NB-DNJ), prevented OC development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of transcriptional activator NFATc1. GM3 was the prevailing GSL produced by patient-derived myeloma cells and MM cell lines, and exogenous addition of GM3 synergistically enhanced the ability of the pro-osteoclastogenic factors RANKL and insulin-like growth factor 1 (IGF-1) to induce osteoclastogenesis in precursors. In WT mice, administration of GM3 increased OC numbers and activity, an effect that was reversed by treatment with NB-DNJ. In a murine MM model, treatment with NB-DNJ markedly improved osteolytic bone disease symptoms. Together, these data demonstrate that both tumor-derived and de novo synthesized GSLs influence osteoclastogenesis and suggest that NB-DNJ may reduce pathological OC activation and bone destruction associated with MM.

Anti-CD138-targeted interferon is a potent therapeutic against multiple myeloma.

Multiple myeloma (MM), a plasma cell malignancy, is the second most prevalent hematologic malignancy in the US. Although much effort has been made trying to understand the etiology and the complexities of this disease with the hope of developing effective therapies, MM remains incurable at this time. Because of their antiproliferative and proapoptotic activities, interferons (IFNs) have been used to treat various malignancies, including MM. Although some success has been observed, the inherent toxicities of IFNs limit their efficacy. To address this problem, we produced anti-CD138 antibody fusion proteins containing either IFNα2 or a mutant IFNα2 (IFNα2(YNS)) with the goal of targeting IFN to CD138-expressing cells, thereby achieving effective IFN concentrations at the site of the tumor in the absence of toxicity. The fusion proteins inhibited the proliferation and induced apoptosis of U266, ANBL-6, NCI-H929, and MM1-144 MM cell lines. The fusion proteins decreased the expression of IFN regulatory factor 4 (IRF4) in U266. In addition, the fusion proteins were effective against primary cells from MM patients, and treatment with fusion proteins prolonged survival in the U266 murine model of MM. These studies show that IFNα antibody fusion proteins can be effective novel therapeutics for the treatment of MM.

Combined immune checkpoint protein blockade and low dose whole body irradiation as immunotherapy for myeloma.

BackgroundMultiple myeloma is characterized by the presence of transformed neoplastic plasma cells in the bone marrow and is generally considered to be an incurable disease. Successful treatments will likely require multi-faceted approaches incorporating conventional drug therapies, immunotherapy and other novel treatments. Our lab previously showed that a combination of transient lymphodepletion (sublethal whole body irradiation) and PD-1/PD-L1 blockade generated anti-myeloma T cell reactivity capable of eliminating established disease. We hypothesized that blocking a combination of checkpoint receptors in the context of low-dose, lymphodepleting whole body radiation would boost anti-tumor immunity.MethodsTo test our central hypothesis, we utilized a 5T33 murine multiple myeloma model. Myeloma-bearing mice were treated with a low dose of whole body irradiation and combinations of blocking antibodies to PD-L1, LAG-3, TIM-3, CD48 (the ligand for 2B4) and CTLA4.ResultsTemporal phenotypic analysis of bone marrow from myeloma-bearing mice demonstrated that elevated percentages of PD-1, 2B4, LAG-3 and TIM-3 proteins were expressed on T cells. When PD-L1 blockade was combined with blocking antibodies to LAG-3, TIM-3 or CTLA4, synergistic or additive increases in survival were observed (survival rates improved from ~30% to >80%). The increased survival rates correlated with increased frequencies of tumor-reactive CD8 and CD4 T cells. When stimulated in vitro with myeloma cells, CD8 T cells from treated mice produced elevated levels proinflammatory cytokines. Cytokines were spontaneously released from CD4 T cells isolated from mice treated with PD-L1 plus CTLA4 blocking antibodies.ConclusionsThese data indicate that blocking PD-1/PD-L1 interactions in conjunction with other immune checkpoint proteins provides synergistic anti-tumor efficacy following lymphodepletive doses of whole body irradiation. This strategy is a promising combination strategy for myeloma and other hematologic malignancies.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-014-0043-z) contains supplementary material, which is available to authorized users.

Effects of Cabozantinib Alone and in Combination with Bortezomib in the 5TGM1 Murine Multiple Myeloma Model

Introduction: Cabozantinib (cabo) is an inhibitor of tyrosine kinases including MET, VEGFR2, RET, and the TAM family kinases TYRO3, AXL, and MER. Cabo has shown clinical activity in patients with castration-resistant prostate cancer and other solid tumors with bone metastases. Multiple myeloma (MM) is the second most common hematologic malignancy, and represents ~2% of all cancer deaths. MM is a monoclonal B-cell (plasma cell) neoplasia with clinical hallmarks of multiple osteolytic lesions causing bone pain, pathologic fractures, and hypercalcemia. Circulating levels of HGF and VEGF are upregulated in MM patients, and regulation of plasma cell-osteoblast communication by the HGF-MET signaling pathway has been implicated in the development of lytic bone disease in these patients. Thus, the primary objectives of this research were to A): determine the activity of cabo on bone lesions and tumor burden in the syngeneic 5TGM1 mouse MM model (study 1), and B): investigate the impact of cabo on overall survival of these mice when dosed alone or in combination with bortezomib (btz) (study 2).Methods: Female C57BL/KaLwRij mice were allocated to treatment groups (n=15-16 per group) with equivalent average body weights. Four experimental groups were utilized in each of 2 studies: a vehicle control group, and groups receiving single agent btz (0.5 mg/kg ip twice a week) or cabo (10 mg/kg, PO QD). Study 1 also included a higher dose cabo group (30 mg/kg, PO QD), and study 2 included a combination group: btz (0.5 mg/kg ip twice a week) plus cabo (10 mg/kg, PO QD). In study 2, each single agent group also received the vehicle from the alternate single-agent group via the appropriate route and schedule.On day 0, animals were inoculated with 5TGM1 mouse myeloma cells by IV administration. Dosing began on day 1 and continued daily until euthanasia at day 35 (study 1) or day 70 (study 2). Body weights were determined twice a week and blood samples were collected on days -1, 15, 22, and 34 for analysis of paraprotein (IgG2b) and TRACP 5b. In study 1 the development of osteolytic lesions was detected by radiography at the end of the study. Some animals were euthanized before the end of the experiment due to paraplegia. Animals euthanized within four days of the end of the experiment in study 1 were included in the analyses.Results: In study 1, btz reduced serum IgG2b levels, and decreased the frequency of soft tissue lesions, but did not show bone protective properties. Cabo exhibited bone protective effects: mean and total area of osteolytic lesions were reduced at the 30 mg/kg dose, and serum TRACP 5b values and osteoclast counts at the tumor-bone interface were reduced at both the 10 and 30 mg/kg doses. Relative bone area did not differ from control according to histomorphometry. The rise in serum IgG2b started earlier than vehicle control in both cabo-treated groups, but a significant difference was not observed in relative IgG2b at sacrifice. Cabo dose dependently increased the necrotic tumor area in bone, indicating the possibility that the rise in IgG2b may have been due to lysis of plasma cells. Both doses of cabo decreased the frequency of soft tissue lesions.In study 2, the median survival times were 36 d (vehicle), 43 d (btz), 48 d (cabo), and 55 d (cabo+btz). The prolongation of OS compared to vehicle was statistically significant for the cabo group but not for the btz group. Prolongation of OS in the combination group was significant compared to btz alone, but not when compared to cabo alone.Conclusions: Cabo showed both bone-protective and anti-tumor effects in this murine model of MM. In addition, statistically-significant prolongation of overall survival was observed with the combination of cabo+btz compared to btz alone, and with single-agent cabo compared to vehicle. Based on these results, further investigation of cabozantinib alone or in combination with other agents in multiple myeloma is warranted. DisclosuresAftab:Exelixis: Employment, Equity Ownership. Suominen:Pharmatest: Employment; Exelixis: Research Funding. Clary:Exelixis: Employment, Equity Ownership. Käkönen:Pharmatest: Employment; Exelixis: Research Funding. Fagerlund:Pharmatest: Employment; Exelixis: Research Funding. Alhoniemi:Pharmatest: Employment; Exelixis: Research Funding. Rissanen:Pharmatest: Employment; Exelixis: Research Funding. Halleen:Pharmatest: Employment; Exelixis: Research Funding.

Liver Macrophages (Kupffer cells) As Novel Targets of Erythropoietin

Erythropoietin (EPO) synthesized by the kidney and by the fetal liver is the major hormone that drives mammalian erythropoiesis. It is thus used in treatment of anemia, associated with chronic renal failure, as well as for alleviating chemotherapy or radiotherapy induced anemia in certain cancer patients. The physiological role of EPO is to regulate production of red blood cells via its receptor, EPO-R. However, EPO-R was found also in various other cell types, as shown also by us, suggesting that EPO may have pleiotropic activities. Originally, we have shown EPO effects on bone-marrow derived macrophages. We now report on EPO effects on liver macrophages. Using transgenic mice, overexpressing human EPO (tg6 mice), we found that they have a 2 fold increased level of F4/80 positive liver macrophages, compared to wt mice (p=0.01). In addition, C57bl/6 wt mice, injected 3 times a week with 180 U/ml of recombinant human EPO (rHuEPO) displayed a 2 fold increase in the percentage of F4/80 expressing cells (p=0.02), and in double positive F4/80 and CD11b expressing cells (p=0.03), compared to their untreated littermates. Similar results were observed in an experimental murine model of multiple myeloma (5T33). Hence, 5T33 MM mice that were treated with rHuEPO (10 consecutive days of 30 U/ml followed by 3 weekly injections of 30 U/ml for 2 weeks), presented a 2.5 fold increase (p=0.0004) and a 3 fold increase (p<0.0001) in the percentage of cells positive for F4/80 and in the percentage of cells double positive for F4/80 and CD11b, respectively. In addition, these mice displayed a 20% increase in the mean fluorescence intensity of CD11b, indicating that EPO causes not only an increase in the proportion of Kupffer cells, but it also induces the activation of CD11b positive cells, as reflected by the elevated expression of this marker per cell. We thus questioned whether Kupffer cells are direct targets of EPO. Our results show that the rat Kupffer cell line (KCL3-2) expresses EPO-R mRNA and protein on the cell surface, as detected by the novel EPO-R antibody, GM1012 generated by the FP7-Health European commission EpoCan grant (Maxwell et al., British Journal of Haematology, accepted for publication). Following treatment of the cells with EPO, the levels of EPO-R mRNA increased by 1.5 fold (p=0.02) and surface EPO-R levels decreased by 2 fold (p≤0.01). Furthermore, stimulation of the KCL3-2 cells with EPO led to activated MAPK signaling, and induced a 50% increase (p<0.005) in cell migration. A 3 fold increase (p=0.04) in the levels of the CCL-2 chemokine mRNA following treatment with EPO, points to a possible mechanism by which monocytes are recruited to the liver, where they differentiate into liver macrophages. Our findings thus introduce and elucidate a new function of EPO, focused on the liver, and indicate its effects in normal as well as in pathological conditions.The first two authors contributed equally to this study.This work was supported by the FP7-Health European commission EpoCan grant (282551). DisclosuresNo relevant conflicts of interest to declare.

Macrophage Migration Inhibitory Factor Regulates Multiple Myeloma Bone Marrow Homing

Multiple myeloma (MM) is the most frequently occurring bone cancer and is characterized by malignant antibody-producing cell accumulation in bone marrow (BM). MM is still incurable, and the disease management is complicated by crosstalk between MM cells and the BM microenvironment. Why MM cells predominantly locate in bone is not well understood. The process by which MM cells are recruited into BM and reside in bone for colonization is called MM BM homing. BM homing is also critical for MM metastasis to distal BM sites. Therefore, MM BM homing is an active process throughout the disease pathogenesis. Here for the first time, we report that macrophage migration inhibitory factor (MIF) regulates MM BM homing via a mechanism that involves MM adhesion in the BM microenvironment. More importantly, we also show that MIF might be a promising target for MM treatment.MIF is an inflammatory cytokine, secreted by various human cell types. Our results here showed that BM biopsies from MM patients had significantly higher MIF expression, compared with that from healthy donors as determined by both immunohistochemistry of BM biopsies and ELISA of BM aspirates (P<0.05). Animal studies using a human MM SCID mouse model (ARP-1 in the SCID mouse) and a murine MM mouse model (5-TGM1 in the C57BL/KawRij mouse) showed that intravenously inoculated (IV) MIF knocked-down (MIF-KD) MM cells did NOT form tumors in bone, whereas control knocked-down cells did form tumors in bone, as determined by both in vivo bioluminescent assay and FACS analysis of BM cells. Unlike the previously identified SDF-1/CXCR4 axis, which regulates MM chemotaxis to BM, MIF knock-down did NOT affect MM cell migration to BM. However, MIF-KD MM cells showed decreased adhesion to BM stromal cells. A mechanistic study suggested that MIF regulation of MM BM homing was mediated by MIF and its receptor CXCR4, but was independent of its primary receptor CD74. The MIF/CXCR4 axis regulated a panel of adhesion molecules via NFκB and JNK intracellular signaling, and MIF-KD and CXCR4-KD MM cells had similar downstream cell signaling and adhesion molecule expression. In addition, exposing MM cells to a MIF inhibitor (4-IPP) down-regulated expression of those adhesion molecules. Further, in the murine MM model with established MM tumors in bone, administering 4-IPP resulted in extramedullary metastasis. Overall, the findings above suggest that MIF regulates MM cell homing to the BM.We also explored MIF-targeting therapy for MM treatment. In the human MM SCID model, IV-inoculated ctrl-KD MM cells formed tumors both in BM and outside of the bone, such as in the abdomen and lung. When tumor-bearing mice were treated with the chemotherapeutic melphalan, the extramedullary tumors regressed significantly (P<0.05), whereas the BM tumors were not sensitive to treatment, probably because the BM microenvironment conferred MM chemoresistance. In the same mouse model inoculated with MIF-KD MM cells, because the MIF-KD MM cells had impaired attachment and could NOT form tumors in bone, the tumor-bearing mice had a complete response to the drug treatment as determined by tumor burden and mouse survival (P<0.05). These encouraging results suggested that MIF might be a valid target for MM treatment. To test this hypothesis, we used the murine MM mouse model and treated the tumor-bearing mice with both melphalan and MIF inhibitor. This combined therapy significantly repressed the MM tumor growth in vivo and lengthened survival (P<0.05). Thus, MIF might be a promising target for MM treatment, in combination with conventional chemotherapy.To summarize, our findings suggest a novel function for MIF in MM BM homing regulation. MIF regulates a panel of adhesion molecules and promotes MM cell attachment within the BM. MIF inhibition disrupts the boundary between MM cells and the BM microenvironment and may raise MM cell drug sensitivity. DisclosuresNo relevant conflicts of interest to declare.

Constitutive gp130-JAK-STAT3 Signaling Induces a Plasma Cell Disorder Representing Genetic and Clinical Features of Human Myeloma – a Novel in Vivo Model for the Patient Subgroup Characterized By a STAT3 Target Gene Signature

Multiple myeloma (MM) is a plasma cell neoplasm characterized by the clonal expansion of an immunoglobulin-secreting terminally differentiated B cell. MM patients classically suffer from the triade consisting of plasma cell infiltration of the bone marrow, lytic bone lesions and monoclonal gammopathy in plasma and/or urine. During disease progress patients develop end organ damage such as nephropathy, neuropathy and bone marrow insufficiency. New treatment options including immunomodulatory drugs and proteasome inhibitors have prolonged overall survival and quality of life. However MM is still considered an incurable disease by conventional treatment approaches.Deregulated signal transduction pathways, in particular the Interleukin-6-JAK-STAT3 pathway, play an important role in the growth regulation and survival of MM cells. IL-6 binds to the specific IL-6 receptor and this complex associates with two molecules of the ubiquitously expressed common signal transducer gp130, which leads to JAK-STAT3 activation and STAT3 target expression. Using immunohistochemistry for STAT3 phosphorylation and by assessing gene expression data we have identified a recurrent STAT3 pathway activation pattern in approximately 40% of all MM patients, strongly indicating that deregulated gp130 downstream activation constitutes an attractive therapeutic target in MM. Based on these findings we generated a novel retroviral bone marrow transduction-transplantation MM mouse model using a constitutively active form of gp130, L-gp130. This L-gp130 mouse model shows all characteristics of the human disease, including monoclonal gammopathy, bone marrow infiltration with lytic bone lesions, and protein deposition in the kidney. Essentially, as compared to previous MM models, MM penetrance is very high and tumor latency with a median of approximately 200 days short. The disease is easily transplantable into secondary recipient mice. To assess clonality we subcloned and sequenced IgH rearrangements from individual tumors. Our analyses revealed that diseased mice contained monoclonal and oligoclonal B cell expansions, and also demonstrated a germinal center passage. Fluorescence in situ hybridization revealed the occurrence of Myc aberrations, a genetic feature that is associated with aggressive disease and short survival in human MM. A genetic in vivo approach revealed that gp130 signaling collaborates with Myc to induce MM, and that gp130 signaling is responsible and sufficient for directing the plasma cell phenotype and to dominantly regulate the expression of antiapoptotic proteins. Finally, testing of murine primary MM cells isolated from diseased L-gp130 animals with currently used anti-myeloma drugs such as bortezomib, carfilzomib, melphalan and doxorubicin revealed significant anti-tumor activity.In summary, the L-gp130 MM mouse model provides a novel tool with clinical and genetic features of human MM. It offers several clear advantages over existing murine MM models. Regarding the patient subgroup with a recurrent STAT3 pathway activation pattern it may serve as a genetic and preclinical tool for delineating MM pathogenesis, for evaluation of targeted therapies, and for assessment of in vivo treatment resistance. DisclosuresKeller:Cellgene: Research Funding.

Surgical Control/Cure of 5TGM1 Murine Multiple Myeloma Model By Thyroparathyroidectomy

The 5TGM1 transplanted C57BL6/KaLwRij mouse is widely used to study murine multiple myeloma development. It recapitulates many features of human multiple myeloma including monoclonal paraprotein as well as bone lesions. Using this mouse model we demonstrated the crucial role of the parathyroid hormone 1 receptor (PTH1R) on the survival of mice exposed to proteasome inhibitor drugs such as bortezomib and carfilzomib; specifically the blockage of the PTH1R function by parathyroid hormone antagonist (PTH 7-34) resulted in significant abrogation of the beneficial survival effect of proteasome inhibitor drugs. To further investigate the effect of the parathyroid hormone (PTH) axis on murine myeloma development we performed thyroparathyroidectomy on the C57BL6/KaLwRij mouse before or after 5TGM1 transplantation.Thyroparathyroidectomy was performed by trained personnel in general anesthesia with isoflurane; all surgical/postsurgical procedures and animal welfare followed an IACUC approved protocol at the University of Arkansas. To prevent hypocalcaemia at postsurgery, 1M CaCl2 solution in the drinking bottle was supplemented for a week. All tested mice received 0.5X106 5TGM1 cell infusion by intravenous injection; mice were divided into three groups; Group 1 control mice (n=10) received 5TGM1 infusion at day 0; Group 2 included 15 mice which on day 10 post 5TGM1 cell infusion underwent thyroparathyroidectomy; Group 3 included 15 mice which at 20 days post-surgery received infusion of 5TGM1 cells. 30% of animals experienced post-surgical complications and died in the first 7 days from procedure; those animals were not included in this survival analysis which is based on 10 animals for each cohort. Blood samples were collected at weekly intervals for IgG2 measurements. The survival curves of mice recovered after surgery are shown in the figure. [Display omitted] All control mice (group 1) developed myeloma progression and died within 3 weeks post-transplant; 2 mice from group 2 and 5 mice of the third cohort manifested disease. All 7 animal deaths were pathologically and serologically attributed to myeloma progression. Compared to controls thyroparathyroidectomized mice (Group 2 and Group 3) showed significantly longer survival p<0.001 and p=0.003, respectively. The 13 surviving mice are still alive 6 months post-procedure and have not shown any serological or clinical evidence of disease progression. Two mice from previous experiment set are still alive and healthy after more than 2 years follow-up. In conclusion thyroparathyroidectomy achieves durable control and possibly cure of the 5TGM1 murine myeloma model suggesting an endocrinological control of myeloma progression. Currently the mechanism of action is under investigation. DisclosuresZangari:Norvartis: Membership on an entity’s Board of Directors or advisory committees; Onyx: Research Funding; Millennium: Research Funding.

Calmodulin antagonists induce cell cycle arrest and apoptosis in vitro and inhibit tumor growth in vivo in human multiple myeloma.

BackgroundHuman multiple myeloma (MM) is an incurable hematological malignancy for which novel therapeutic agents are needed. Calmodulin (CaM) antagonists have been reported to induce apoptosis and inhibit tumor cell invasion and metastasis in various tumor models. However, the antitumor effects of CaM antagonists on MM are poorly understood. In this study, we investigated the antitumor effects of naphthalenesulfonamide derivative selective CaM antagonists W-7 and W-13 on MM cell lines both in vitro and in vivo.MethodsThe proliferative ability was analyzed by the WST-8 assay. Cell cycle was evaluated by flow cytometry after staining of cells with PI. Apoptosis was quantified by flow cytometry after double-staining of cells by Annexin-V/PI. Molecular changes of cell cycle and apoptosis were determined by Western blot. Intracellular calcium levels and mitochondrial membrane potentials were determined using Fluo-4/AM dye and JC-10 dye, respectively. Moreover, we examined the in vivo anti-MM effects of CaM antagonists using a murine xenograft model of the human MM cell line.ResultsTreatment with W-7 and W-13 resulted in the dose-dependent inhibition of cell proliferation in various MM cell lines. W-7 and W-13 induced G1 phase cell cycle arrest by downregulating cyclins and upregulating p21cip1. In addition, W-7 and W-13 induced apoptosis via caspase activation; this occurred partly through the elevation of intracellular calcium levels and mitochondrial membrane potential depolarization and through inhibition of the STAT3 phosphorylation and subsequent downregulation of Mcl-1 protein. In tumor xenograft mouse models, tumor growth rates in CaM antagonist-treated groups were significantly reduced compared with those in the vehicle-treated groups.ConclusionsOur results demonstrate that CaM antagonists induce cell cycle arrest, induce apoptosis via caspase activation, and inhibit tumor growth in a murine MM model and raise the possibility that inhibition of CaM might be a useful therapeutic strategy for the treatment of MM.

Abstract 734: Effects of Cabozantinib in the 5TGM1 murine multiple myeloma model

Abstract Cabozantinib is an inhibitor of tyrosine kinases including MET, VEGFR2, and RET, and has shown clinical activity in patients with castration-resistant prostate cancer and other solid tumors with bone metastases. Multiple myeloma (MM) is the second most common hematologic malignancy, and represents ∼2% of all cancer deaths. MM is a monoclonal B-cell (plasma cell) neoplasia with clinical hallmarks of multiple osteolytic lesions causing bone pain, pathologic fractures, and hypercalcemia. Circulating levels of HGF and VEGF are upregulated in MM patients, and regulation of plasma cell-osteoblast communication by the HGF-MET signaling pathway has been implicated in the development of lytic bone disease in these patients. Thus, our aim was to determine the activity of cabozantinib on bone lesions and tumor burden in the syngeneic 5TGM1 mouse MM model. Four experimental groups were included: negative control group receiving vehicle, positive control group receiving bortezomib (0.5 mg/kg ip twice a week), low dose cabozantinib group (10 mg/kg, PO QD) and high dose cabozantinib group (30 mg/kg, PO QD). Female C57BL/KaLwRij mice were allocated to treatment groups (n=15 per group) with equivalent average body weights. On day 0, animals were inoculated with 5TGM1 mouse myeloma cells by IV administration. Dosing began on day 1 and continued daily until euthanasia at day 35. Body weights were determined twice a week and blood samples were collected on days -1, 15, 22, and 34 for analysis of paraprotein (IgG2b) and TRACP 5b. The development of osteolytic lesions was detected by radiography at the end of the study. Some animals (4/15) were euthanized before the end of the experiment due to paraplegia in control and bortezomib groups, but none in cabozantinib groups. Animals euthanized within four days of the end of the experiment were included in the analysis. Bortezomib reduced serum IgG2b levels and decreased the frequency of soft tissue lesions, but did not show bone protective properties. Cabozantinib exhibited bone protective effects: mean and total area of osteolytic lesions were reduced at the 30 mg/kg dose, and serum TRACP 5b values and osteoclast counts at the tumor-bone interface were reduced at both the 10 and 30 mg/kg doses. Relative bone area did not differ from control according to histomorphometry. The rise in serum IgG2b started earlier than vehicle control in both cabo-treated groups, but a significant difference was not observed in relative IgG2b at sacrifice. Cabozantinib dose dependently increased the necrotic tumor area in bone, indicating the possibility that the rise in IgG2b may have been due to lysis of plasma cells. Both cabozantinib doses decreased the frequency of soft tissue lesions. In summary, cabozantinib showed both bone-protective and anti-tumor effects in this murine model of MM. Based on these promising results, further investigation of cabozantinib in multiple myeloma is warranted. Citation Format: Mari I. Suominen, Douglas O. Clary, Rami Käkönen, Katja M. Fagerlund, Esa Alhoniemi, Jukka P. Rissanen, Jussi M. Halleen, Dana T. Aftab. Effects of Cabozantinib in the 5TGM1 murine multiple myeloma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 734. doi:10.1158/1538-7445.AM2014-734

Transmissible cytotoxicity of multiple myeloma cells by cord blood-derived NK cells is mediated by vesicle trafficking.

Natural killer cells (NK) are important effectors of anti-tumor immunity, activated either by the downregulation of HLA-I molecules on tumor cells and/or the interaction of NK-activating receptors with ligands that are overexpressed on target cells upon tumor transformation (including NKG2D and NKP30). NK kill target cells by the vesicular delivery of cytolytic molecules such as Granzyme-B and Granulysin activating different cell death pathways, which can be Caspase-3 dependent or Caspase-3 independent. Multiple myeloma (MM) remains an incurable neoplastic plasma-cell disorder. However, we previously reported the encouraging observation that cord blood-derived NK (CB-NK), a new source of NK, showed anti-tumor activity in an in vivo murine model of MM and confirmed a correlation between high levels of NKG2D expression by MM cells and increased efficacy of CB-NK in reducing tumor burden. We aimed to characterize the mechanism of CB-NK-mediated cytotoxicity against MM cells. We show a Caspase-3- and Granzyme-B-independent cell death, and we reveal a mechanism of transmissible cell death between cells, which involves lipid-protein vesicle transfer from CB-NK to MM cells. These vesicles are secondarily transferred from recipient MM cells to neighboring MM cells amplifying the initial CB-NK cytotoxicity achieved. This indirect cytotoxicity involves the transfer of NKG2D and NKP30 and leads to lysosomal cell death and decreased levels of reactive oxygen species in MM cells. These findings suggest a novel and unique mechanism of CB-NK cytotoxicity against MM cells and highlight the importance of lipids and lipid transfer in this process. Further, these data provide a rationale for the development of CB-NK-based cellular therapies in the treatment of MM.

A novel TLR-9 agonist C792 inhibits plasmacytoid dendritic cell-induced myeloma cell growth and enhance cytotoxicity of bortezomib

Our prior study in multiple myeloma (MM) patients showed increased numbers of plasmacytoid dendritic cells (pDCs) in the bone marrow (BM), which both contribute to immune dysfunction as well as promote tumor cell growth, survival and drug resistance. Here we show that a novel Toll-like receptor (TLR-9) agonist C792 restores the ability of MM patient-pDCs to stimulate T-cell proliferation. Coculture of pDCs with MM cells induces MM cell growth; and importantly, C792 inhibits pDC-induced MM cell growth and triggers apoptosis. In contrast, treatment of either MM cells or pDCs alone with C792 does not affect the viability of either cell type. In agreement with our in vitro data, C792 inhibits pDC-induced MM cell growth in vivo in a murine xenograft model of human MM. Mechanistic studies show that C792 triggers maturation of pDCs, enhances interferon-α and interferon-λ secretion and activates TLR-9/MyD88 signaling axis. Finally, C792 enhances the anti-MM activity of bortezomib, lenalidomide, SAHA or melphalan. Collectively, our preclinical studies provide the basis for clinical trials of C792, either alone or in combination, to both improve immune function and overcome drug resistance in MM.

Tumor Suppressor Microrna-29a/b and Microrna-34a Mediate Small Molecule PRIMA-1Met-Induced Aoptosis In Multiple Myeloma Cells By Targeting c-Myc

PRIMA-1Met/APR246 (p53 reactivation and induction of massive apoptosis), is a small molecule with remarkable anti-tumor activities in various human tumor cells, and is currently under phase I/II clinical trial. We have previously demonstrated anti-tumor activity of PRIMA-1Met in multiple myeloma (MM) cells irrespective of p53 status. In addition, we have shown that PRIMA-1Met alone or in combination with dexamethasone triggers significant tumor growth inhibition in vivo in a murine xenograft model of human MM. However, the molecular mechanism underlying anti-myeloma activity of PRIMA-1Met has not been fully elucidated. MicroRNAs (miRNAs) are non-coding small RNA molecules that regulate post-transcriptional gene expression and play a critical role in tumor pathogenesis. Since the role of miRNAs and their regulation in response to PRIMA-1Met in MM is not known, here we investigated the relationship between PRIMA-1Met-induced apoptosis and miRNA expression in MM cells. Using a miRNA PCR array platform (Human Cancer Pathway Finder miScript miRNA PCR array, MIHS-102Z, Qiagen Inc), we analyzed the miRNA profiles in two MM cell lines of different p53 status (MM.1S with wild type p53 and 8226 with mutant p53) treated with either PRIMA-1Met or DMSO control. After normalization to a set of housekeeping genes, differential expressions of the miRNAs were analysed. miRNA-29a, miRNA-29b, and miRNA-34a were found significantly up-regulated (more than 2 fold, p<0.05) in cells treated with PRIMA-1Met compared to DMSO-treated cells. To evaluate the effect of over-expression of these miRNAs, we transfected two MM cell lines (MM.1S and 8226) with either miR-29a/b or miR-34a. Cells transfected with scramble miRNA were used as control. Over-expression of the miRNAs resulted in a dose-dependent inhibition of viability and increase in apoptosis of MM.1S or 8226 cells. Next, we examined the endogenous expression of these miRNAs in 5 primary MM samples by qPCR. Results showed a significant low expression of miR-29a/b and miR-34a in 3 of the 5 samples. Treatment of the two primary MM samples with low expression for miR-29a/b and miR-34a with PRIMA-1Met resulted in up-regulation of these miRNAs leading to inhibition of the viability and induction of apoptosis. To identify the possible targets of these miRNAs, we performed bioinformatics analysis. Results obtained from different searches by miRanda and TargetScan algorithm predicted c-Myc as a potential target for miRNA-29a/b and miRNA-34a. c-Myc is an oncogene whose over-expression has been associated with resistance to current chemotherapy in MM. Global gene expression profiling by microarray showed significant down-regulation of c-Myc in two MM cell lines with either wild type or mutant p53 treated with PRIMA-1Met compare to cells treated with DMSO. Importantly, down-regulation of c-Myc (∼2.6-fold) by PRIMA-1Met was also observed in a MM cell line (8226R5) lacking p53 expression suggesting an important role of c-Myc in p53-independent apoptosis of MM cells induced by PRIMA-1Met. By qPCR and Western blot analysis, we confirmed significant down-regulation of c-Myc in PRIMA-1Met-treated MM cells. These data provided the evidence for an inverse correlation between the expression of these miRNAs and c-Myc indicating that apoptosis of MM cells induced by PRIMA-1Met is regulated by miRNAs29a/b or miRNA34a targeting c-Myc. Our results suggest a novel mechanism for PRIMA-1Met-induced apoptotic signaling in MM cells mediated by up-regulation of miR-29a/b and miR-34a targeting c-Myc. Our findings also provide a preclinical framework for development of therapeutic strategies in combination of PRIMA-1Met and miRNA (miR-29a/b or miR-34a) mimics for the treatment of MM patients, especially for those with high c-Myc expressions. Disclosures:No relevant conflicts of interest to declare.

Antitumor Activities Of An Oral Selective HSP90α/β Inhibitor, TAS-116, In Combination With Bortezomib In Multiple Myeloma

Heat-shock protein 90 (HSP90) is a molecular chaperone considered to be attractive therapeutic target in many cancers, including multiple myeloma (MM), since HSP90 inhibition triggers downregulation /degradation of numerous client proteins to trigger apoptosis. In this study, we examined the anti-MM activities of a novel and orally bioavailable HSP90α/β inhibitor TAS-116 (TAIHO PHARMACEUTICAL CO., LTD., Japan) in MM. TAS-116 triggered downregulation of known client proteins associated with significant growth inhibition in MM cell lines and patient MM, without cytotoxicity in normal peripheral blood mononuclear cells. Importantly, TAS-116 showed cytotoxicity even in 17-allylamino-17-demethoxygeldanamycin (17-AAG)-resistant acute B cell leukemia cell line NALM6. TAS-116 induced cell cycle arrest, associated with downregulation of cell cycle regulatory proteins; followed by apoptosis, evidenced by increased annexin V-positive staining, as well as cleavage of caspases and PARP. Since the bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis including drug resistance, we further examined the effect of TAS-116 in the presence of BM stromal cells (BMSCs). TAS-116 induced significant cytotoxicity in MM cells even in the presence of BMSCs, associated with downregulation of phospho-Akt and phospho-ERK. In addition, neither exogenous IL-6 nor IGF-1 was able to protect against TAS-116. Importantly, TAS-116 did not induce cytotoxicity in BMSCs. Our previous studies have shown that combined HSP90 inhibitor and proteasome inhibitor treatment induces synergistic MM cell death; therefore we similarly examined whether TAS-116 enhances bortezomib-induced cytotoxicity. TAS-116 enhanced bortezomib-induced cytotoxicity in MM.1S and RPMI-8226 cells, associated with decreased phospho-Akt, phospho-RelA (p65), phospho-IkBa, and phospho-IKKα/β; as well as increased CHOP and PARP cleavage. These results suggest that TAS-116 blocks bortezomib-induced canonical NF-κB pathway and augments bortezomib-induced endoplasmic reticulum (ER) stress. Finally, we examined the in vivo efficacy of TAS-116 in combination with bortezomib using a xenograft murine model of human MM. Although both monotherapy of TAS-116 and bortezomib were effective, TAS-116 in combination with bortezomib significantly enhanced growth inhibition (80%, p&lt;0.05) and prolonged overall survival (29 vs 57 days, p&lt;0.001). Immunohistochemical analysis of harvested human MM confirmed a significant increase in cleaved caspase-3- and TUNEL-positive cells in TAS-116 animals. These treatments were well tolerated, and no significant body weight loss of the animals was observed. Taken together, our studies show that the novel and selective HSP90α/β inhibitor TAS-116 blocks MM cell growth both in vitro and in vivo, providing the preclinical framework for clinical evaluation of this agent to improve patient outcome in MM. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Ohkubo:TAIHO PHARMACEUTICAL Co.,LTD: Employment. Richardson:Millenium: Consultancy; Celgene: Consultancy; Johnson &amp; Johnson: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Munshi:Celgene: Consultancy; Novartis: Consultancy; Millennium: Consultancy. Utsugi:TAIHO PHARMACEUTICAL CO.,LTD: Employment. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

Nanoparticle Arsenic Compound Realgar Effectively Targets Myeloma Stem-Like Side Population

Introduction Tumors contain diverse sub-clones, which can lead to tumor persistence and re-growth after therapy. Previous studies suggest that multiple myeloma (MM) sub-clones may contain tumor-initiating cells which maintain growth and proliferation as well as contribute to drug-resistance. Recently, we have confirmed the existence of MM stem cell-like cells by identifying “side population” (SP) cells as an enriched source of tumor-initiating cells with clonogenic and tumorigenic stem cell properties. Targeting this population with novel therapies represents a promising strategy. Arsenic trioxide (ATO, As2O3) shows only limited responses compared to anti-MM agents such as bortezomib and lenalidomide in relapsed and refractory MM. Here we evaluated the anti-MM activity of another arsenic compound (arsenic sulfide, As4S4), used in traditional Chinese medicine with greater efficacy and less genotoxicity than ATO, prepared by milling into realgar nanoparticles (NREA). We assessed the effect of NREA on MM stem cell-like SP fraction in the context of the bone marrow stromal cells (BMSCs) to provide the rational for its clinical evaluation. Methods and Results First, we evaluated the effect of both NREA and ATO on MM cell survival. Both drugs significantly decreased survival of 13 MM cell lines in a concentration-dependent manner. Importantly, we observed more potent anti-MM effect with NREA compared to ATO, with IC50 values of NREA 2-4 times lower than ATO. Moreover, we confirmed the effect of NREA on MM cells from patients with primary relapsed/refractory disease: NREA showed a dose-dependent response, with IC50 values in the range 1-2 μM. Significant concentration-dependent increased apoptosis was observed in NREA-treated MM cells. Similarly, decreased mitochondrial membrane potential was more significantly triggered by NREA than ATO. Consequently, NREA modulated the MM cell cycle profile: concentration-dependent treatment induced either G0/G1 (MM.1S and KMS11) or G2/M arrest (RPMI-S and OPM1) at 48h. To compare in vivo anti-tumor activity of NREA and ATO, we used our xenograft murine model of human MM. Tumor-bearing mice were intraperitoneally treated with NREA, ATO or with the respective vehicle. Treatment with NREA triggered more significant tumor growth inhibition compared to ATO at day 8 of treatment. To evaluate the effect of NREA on the SP phenotype of MM cells, we treated MM cells with subtoxic concentrations of NREA and ATO for 72 h. Analysis of MM cells for low intracellular content of Hoechst 33342 (SP fraction) revealed that NREA significantly decreased the percentage of SP cells in MM cells in a dose-dependent manner, whereas increase in SP fraction was observed in ATO-treated cells. To evaluate the effect of the bone marrow microenvironment on targeting the SP cells with NREA, MM cells were cultured alone or with BMSCs, and then analyzed for low intracellular accumulation of Hoechst 33342. Treatment by NREA, but not ATO, significantly depleted SP cells in co-cultures MM cells with BMSCs. Moreover, NREA decreased clonogenic potential, whereas ATO had no effect. Similarly, tumorigenic potential of SP cells was significantly decreased by NREA, but not by ATO, treatment. Conclusions Overall, our results demonstrate significant anti-tumor activity of NREA against MM cells in vitro. Our in vivo data further show that NREA significantly decreased tumor burden at clinically achievable concentrations of arsenic. Importantly, targeting of SP cells by NREA in the context of BMSCs provides the preclinical rationale for its clinical evaluation. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Richardson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Johnson &amp; Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.