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Multiple myeloma (MM), the second most common hematologic malignancy worldwide, is a B cell malignancy characterized by high frequency of intra-clonal diversity within malignant plasma cells (PC) in the bone marrow (BM). To better understand the myeloma heterogeneity within its complex pathophysiology, we performed large-scale data-driven mass cytometry (CyTOF) analysis in cohort of 188 bone marrow (BM) samples from multiple myeloma (MM) patients compared to 10 age-matched healthy donors (HD). Our design focused on profiling of PC intra and inter-neoplastic heterogeneity based on molecular perturbations of transcriptional factors and signaling regulators and stemness-controlling markers ensuring development of B cell lymphopoiesis within myelomagenesis encompassing the different clinical spectra of pre-malignant/asymptomatic (16 MGUS and 25 SMM) and active symptomatic stages (43 NDMM and 104 relapsed or relapsed/refractory MM patients) of MM pathogenesis. Moreover, interaction of PC disease status with the immune ecosystem of myeloma microenvironment was evaluated as well. To distinguish tumor-driven specific immune changes from myeloma immune ecosystem, we observed that cell frequency of cytotoxic naïve and effector cells, g/dT, and early monocytes, myelocytes and erythroblasts immune subsets was significantly reduced in both premalignant and active MM stages. In contrast, mostly innate immune clusters including non-canonical monocytes, myeloblasts, and mature neutrophils, erythroblasts and platelets were present at a higher frequency across all MM stages versus HD. To evaluate cell distribution of B lymphopoiesis in MM disease stages, switched memory B cells and plasmablasts clusters were upregulated in premalignant stage MGUS compared to HD. Similar observations were detected in SMM and NDMM versus HD, with the highest abundance of PC clusters in NDMM. The downregulation of cell distribution in B cell progenies, immature and transitional B cells, and un-switched memory B cell clusters was observed in NDMM and relapsed/refractory MM patients. Furthermore, MM patients treated with Revlimid-Velcade-Dexamethasone therapy had decrease frequency of specific PC clusters and un-switched and transitional B cell clusters. In addition, our data revealed immunophenotyping aberrancies present not only in PC clusters but also across all myeloma B lymphomagenesis in BM samples from MM patients. In-depth characterization of malignant plasma cells, significant variations were detected in PC clusters of MM cohort based on different expression of IRF4, c-Myc, CD28, CD117, and FGFR-3, however with homogenous expression of sXBP1, and MMSET which differ in all 4 MM stages compared to HD. Significant upregulation of CD47 was showed in all PC clusters of MM cohort. Moreover, PC clusters differ in intra-clonal expression of self-renewing/stemness markers CD184, Notch-1, Oct3/4, KLF-4, Sox-2, and Nanog, supporting the idea of sub-clonal variations insight of MM tumor. This study might provide the rational for prediction of MM patient status and design of targeted therapy in MM on personalized bases. This work was supported by REA grant agreement No. 609427-SASPRO 0064/01/02, TRS-2015-00000170, APVV-16-0484 and VEGA 2/0076/17. Disclosures Hunter: Janssen: Consultancy. Jamroziak:Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Richardson:Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding. Kastritis:Prothena: Honoraria; Genesis: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Pfizer: Honoraria. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Waldenström macroglobulinemia (WM), a malignant B-cell lymphoplasmacytic lymphoma, is a rare subtype of non-Hodgkin lymphoma representing about 1% of all cases. To better understand the WM pathogenesis, we performed large-scale data-driven proteomic profile of WM tumor cells associated with tumor-driven immune changes in the tumor microenvironment of 66 bone marrow (BM) samples from WM patients compared to 10 age-matched healthy donors (HD) by time-of-flight mass cytometry (CyTOF) technology. Our workflow has been designed based on extensive 3 CyTOF antibody panels to evaluate WM tumor within B cell lymphopoiesis concurrently with immune landscape of the tumor microenvironment in WM by state-of-art technology CyTOF. To map B cell lymphomagenesis in WM, we defined whole spectrum of maturation of B cell development, from hematopoietic stem cells and B cell precursors through immature B cells, transitional B cells, and naïve B cells together with memory un-switched and switched B cells, plasmablasts and plasma cells in BM samples of WM patients by positive and negative co-expression of 13 B cell-stage specific markers. Various immunophenotyping aberrancies within WM B lymphomagenesis were associated with WM clones characterized by significant increase of 11 B subset clusters from un-switched and switched memory B cells to plasma cells. Interestingly, WM clusters differ in intra-clonal expression of activation surface molecules (CD23, CD24, CD25, CD81, CD329, CD200, and CD319); transcriptional factors and regulators controlling B cell development (MYD88, Bcl-6, IRF-4, sXBP-1, and FGFR-3) and stemness-related markers (Oct3/4, Nanog, Sox-2, c-Myc, and Notch-1) in WM supporting the idea of sub-clonal heterogeneity insight of WM tumor. Moreover, decrease in cell frequency of B cell precursors (pro-B and pre-BI), naive B cells, and plasmablasts were observed in WM patients versus HD. To generate a comprehensive view of the tumor microenvironment, we observed significant upregulation of g/dT cells, CD4+ and CD8+ T effector cells, CD8+ T effector memory cells, monocytes, and neutrophils immune subsets and downregulation of immature T cells, CD8+ T naïve cells, plasmacytoid dendritic cells, myelo/mono progenitor clusters. Ibrutinib (IBRU) treatment has been effective in relapsed/refractory WM patients; therefore highest numbers of WM patients were receiving IBRU therapy in our cohort. IBRU treated WM patients had decreased frequency of naive B, CD4+ T naive cells and specific clusters of un-switched and switched memory B cells. Moreover, responder versus non-responders to IBRU therapy revealed increase of CD8+T effector memory cells. In sum, correspondence analysis reflecting data of each patient and immune subsets revealed stratification of WM patients with reflection on their clinical outcome, therefore providing the rational for prediction of WM patient status. This study was supported by APVV-16-0484 and VEGA 2/0076/17. Disclosures Hunter: Janssen: Consultancy. Richardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Kastritis:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria. Treon:BMS: Research Funding; Janssen: Consultancy; Pharmacyclics: Research Funding. Anderson:Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Recurrence of multiple myeloma (MM) after therapy suggests the presence of tumor-initiating subpopulations. In our study, we performed flow cytometry–based Hoechst 33342 staining to evaluate the existence of a MM population with stem-like features known as side population (SP) cells. SP cells exhibit substantial heterogeneity in MM cell lines and primary MM cells; express CD138 antigen in MM cell lines; display higher mRNA expression and functional activity of ABCG2 transporter; and have a higher proliferation index compared with non-SP cells. We observed evidence for clonogenic potential of SP cells, as well as the ability of SP cells to regenerate original population. Moreover, SP cells revealed higher tumorigenicity compared with non-SP cells. Importantly, lenalidomide decreased the percentage and clonogenicity of SP cells, and also induced phosphorylation changes in Akt, GSK-3α/β, MEK1, c-Jun, p53, and p70S6K in SP cells. Adherence to bone marrow stromal cells (BMSCs) increased the percentage, viability, and proliferation potential of SP cells. Lenalidomide and thalidomide abrogated this stimulatory effect of BMSCs and significantly decreased the percentage of SP cells. Our studies demonstrate a novel mechanism of action for lenalidomide, namely targeting SP fraction, providing the framework for new therapeutic strategies targeting subpopulations of MM cells including presumptive stem cells.

The transformation from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) is thought to be associated with changes in immune processes. We have therefore used serologic analysis of recombinant cDNA expression library to screen the sera of MGUS patients to identify tumor-associated antigens. A total of 10 antigens were identified, with specific antibody responses in MGUS. Responses appeared to be directed against intracellular proteins involved in cellular functions, such as apoptosis (SON, IFT57/HIPPI), DNA and RNA binding (ZNF292, GPATCH4), signal transduction regulators (AKAP11), transcriptional corepressor (IRF2BP2), developmental proteins (OFD1), and proteins of the ubiquitin-proteasome pathway (PSMC1). Importantly, the gene responsible for the oral-facial-digital type I syndrome (OFD1) had response in 6 of 29 (20.6%) MGUS patients but 0 of 11 newly diagnosed MM patients. Interestingly, 3 of 11 (27.2%) MM patients after autologous stem cell transplantations showed responses to OFD1. We have confirmed T-cell responses against OFD1 in MGUS and observed down-regulation of GLI1/PTCH1 and p-β-catenin after OFD1 knock-down with specific siRNA, suggesting its functional role in the regulation of Hh and Wnt pathways. These findings demonstrate OFD1 as an important immune target and highlight its possible role in signal transduction and tumorigenesis in MGUS and MM.

Background: Despite advances in treatment, multiple myeloma (MM) remains incurable due to development of drug resistance in the bone marrow microenvironment. Microtubules (MTs) are dynamic protein biopolymers formed through polymerization of heterodimers of α- and β-tubulins. Disruption of microtubules induces cell cycle arrest in G2-M phase and formation of abnormal mitotic spindles. MTs are also involved in many processes in interphase cells, including intracellular trafficking, cell motility and angiogenesis. The important functions of MT in the cells make them an attractive target for anti-myeloma drug discovery. Furan metotica is a novel class of anti-mitotic spindle drugs that inhibit kinetochore-microtubule binding and trigger a spindle checkpoint mediated arrest in mitosis, which frequently ends in cell death. We evaluated the activity of STK405759, a member of the furan metotica family, as a novel, potential antitubulin drug for MM treatment in preclinical models. Methods: Cytotoxic activity of STK405759 was evaluated by XTT assay. Apoptosis and cell cycle were analyzed by flow cytometry. Tubulin polymerization inhibition was evaluatedusing a biochemical cell free assay and by testingthe levels of soluble and polymerized tubulin in MM-treated cells using Western blot analysis. Efficacy and toxicity of the drug were checked in a murine MM xenograft model. Histochemistry was used to assay tumor apoptosis. Results: STK405759 had a potent cytotoxic activity against a wide variety of MM cell lines and patient-derived MM cells, regardless of their sensitivity to conventional therapy or novel agents. In contrast, the viability of normal peripheral blood mononuclear cells derived from healthy donors and MM patients was not affected. Importantly, STK405759 induced cell death of RPMI MM cells co-cultured with HS-5 bone marrow stromal cells. STK405759 inhibited tubulin polymerizationin a cell free system anddecreased the level of polymerized tubulin in MM treated cells.The STK405759 anti-tubulin activity was supported by demonstration of MM cell cycle arrest followed by activation of an apoptotic default pathway. Activation of pro-caspase-8 and poly (ADP-ribose) polymerase in the cleaved forms, as well as down-regulation of the Mcl-1 anti-apoptotic protein was detected in RPMI treated cells. Combination studies of STK405759 with bortezomib, lenalidomide or dexamethasone showed significant synergistic and additive cytotoxicity in MM cells. In vivo studies revealed decreased MM tumor burden and prolonged survival of STK405759-treated mice compared to controls. STK405759 induced apoptosis of tumors cells from treated mice. Summary/Conclusion: STK405759 is an active, microtubule-targeting agent with potent anti-myeloma activity. These results provide a rationale for further evaluation of STK405759 as monotherapy or part of combination therapy for treating patients with MM. Disclosures No relevant conflicts of interest to declare.

Introduction: CyTOF (time-of-flight mass cytometry) is a novel high-dimensional technology which permits immunophenotyping and analysis of signaling in single cells. This approach enables simultaneous evaluation of up to 40 parameters using antibodies tagged with distinct elemental isotopes, by combining flow cytometry with atomic mass spectrometry. Since multiple myeloma (MM) is characterized by immune dysfunction, we used CyTOF technology to define the complex immune profile in MM patient bone marrow (BM) samples. Methods: We used 40 different markers to define various B, T, natural killer (NK) subsets, as well as cells of monocytic, granulocytic, erythroid and platelet lineages. Our preliminary data are results from 10 patients with MGUS/ smoldering MM (SMM); 10 newly diagnosed MM; 20 relapsed/refractory MM; and 15 WM patients (5 newly diagnosed and 10 receiving treatment) in comparison with age-matched healthy donors’ BM (HD). A significantly larger cohort of MM (N=150) and WM (N=50) patients is being similarly analyzed and will be presented. To evaluate phenotypic abnormalities in various B cell subsets, we used B lineage markers CD10, CD19, CD20, CD22, CD27, CD34, CD38, CD45, IgA, IgD, IgG and IgM to define B cells maturation stages from hematopoietic stem cells (HSC) to naïve to mature B lymphocytes (pro-B; pre-B-I; pre-B-II; immature B; and mature (naïve) B cells), as well as memory non-switched and memory switched B cells, plasmablasts, normal (CD138+CD38+CD19+CD45+) and clonal plasma cells (CD138+CD38+CD19-CD45-/low), which reside in the specific BM niche. Furthermore, natural killer (NK) subsets (such as NK and NKT cells) and T cells (such as memory CD4T, naive CD4T, memory CD8T, naive CD8T, T regulatory cells and Tg/d cells) were examined. High-dimensional data was obtained using CyTOF technology and analyzed by SPADE and viSNE software. Results: Our data showed significantly decreased HSC in patients with newly diagnosed and relapsed/refractory MM compared to HD (P Conclusion: A better understanding of the neoplastic BM milieu will provide the framework for identifying and validating novel targeted therapies directed against MM. CyTOF technology represents a novel diagnostic tool to assess the status not only of MM, but also of host immunity, and may allow for the development of rational personalized therapies. Disclosures No relevant conflicts of interest to declare.

Introduction: Intra-clonal heterogeneity in malignant plasma (PC) cells and B-cells has recently been reported in both multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM). Further phenotypic and molecular characterization of inter- and intra-clonal genetic complexity will enhance our understanding of disease pathogenesis and identify novel therapeutic strategies. Methods: In this study, we compared normal and malignant PC maturation-associated B-cell subsets using bone marrow samples from individuals with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), newly diagnosed MM, and relapsed/refractory MM versus age-matched healthy donors (HD). We also similarly analyzed WM. In addition to corrupted B-cell lineage, we examined phenotypic and molecular features of intra-clonal architecture (complexity) of malignant PC in MM and clonal B-cells in WM on a single cell level using time-of-flight mass cytometry (CyTOF) technology. CyTOF technology is based on rare stable earth elemental isotopes-bound to antibodies to target epitopes on and within cells: up to 40 different markers on a single cell can simultaneously assess including phenotype, transcription factors, regulatory signaling molecules and enzymes, as well as activation of signaling molecules. The resulting high-dimensional data were analyzed by SPADE, viSNE and Wanderlust software. Results: Our high-dimensional data of clustered analyses showed significantly decreased CD19+CD27- patient cells in MM with cytogenetic abnormalities (cytog+) including del(13q), t(4;14), t(14;16), t(3;14), +1q or t(11;14) versus patient cells in MM without any cytogenetic abnormalities (cytog-; P=0.013). In contrast, there was a significant increase of transitional B cells (CD19+CD27-IgM+CD10-IgDlow) in patients with MM cytog+ vs. MM cytog- (P=0.028). A significant increase of mature (naïve) B cells (CD19+CD27-IgM+CD10-IgD+) was also detected in MM cytog+ versus MM cytog- patients (P=0.013), but not in WM cytog- vs. WM cytog+ (46XY, -Y, +18q, +6p, 14q). Clonal PC (CD19-CD38++CD138+CD45-/dim; either cyk or cyl +) were significantly upregulated in MM cytog+ compared to MM cytog- (P=0.021) by CyTOF analyses. To investigate phenotypic profiles and molecular signature of intra-clonal heterogeneity of PC in MM, high-dimensional analyses by SPADE and viSNE revealed that clonal PC clustered separately from B cells by, virtue of high CD319 and CD47 expression; variable expression of CD52, CD56, CD81, CD44, CD200; and low expression of CD28, CD117, CD338, CD325, and CD243. For example, adhesion CD56 and anti-adhesion CD52 molecules were significantly increased in MM cytog+ compared to MM cytog-. Clonal PC highly expressed IFR4 and Notch1; variously expressed FGFR3, sXBP-1, KLF4 and c-Myc; and only minimally expressed Bcl-6, WHSC-1 (MMSET) and RARa2. sXBP-1 was significantly upregulated in all MM stages compared to HD. Furthermore, expression of stem cell markers including Sox-2, Oct3/4 and Nestin was detected only at low level in clonal PC, except for higher expression of Nanog. In WM, clonal B cells expressed Bcl-6 (4-36%) and MYD88 (2-27%) by CyTOF analyses. Finally, cluster analyses by SPADE and viSNE allows for detection of phenotypic and molecular changes not only in clonal populations but also at distinct B-lineage maturation stages, such as expression of Pax-5 and Bcl-2 on early B cell progenitors. This data represents a cohort of MM (N=35) and WM (N=15) patients; a significantly larger data set of MM (N=100) and WM (N=50) will be presented. Conclusion: This study characterizes the molecular and phenotypic profile associated with inter- and intra-clonal heterogeneity in MM and WM. It not only enhances our understanding of disease pathogenesis, but may allow for individualized targeted therapy. Disclosures No relevant conflicts of interest to declare.

Introduction: Recent studies have elucidated the importance of using 3-dimensional rather than 2-dimensional models in order to create an experimental system recapitulating the specialized properties of the bone marrow microenvironment. Since the neoplastic bone marrow (BM) milieu plays important roles in multiple myeloma (MM) pathogenesis, novel models to study the MM cell in its neoplastic microenvironment are needed. Methods: To mimic the neoplastic BM microenvironment of MM patients, we have established a special hydrogel-based 3-dimensional (3-D) model by ex-vivo culturing MM patient-derived mesenchymal stem cells (MM-MSCs), the predominant cellular component of the marrow niche, which promotes greater mineralization and differentiation than a 2-dimensional (2-D) system. Results: To characterize MM-MSCs in different stages of MM, we utilized an 11 multi-color flow cytometry panel. The percentage of MSCs (CD73+CD90+CD105+lin-CD45-CD34-HLA-DR-) population in BM aspirate samples of 50 MM patients (MGUS, smoldering MM, newly diagnosed MM, and relapsed or relapsed/refractory MM) was evaluated, and correlated with the distribution of (CD38+ CD138+) plasma cells. MSCs were less frequent (10x) than plasma cells, and increased with disease progression to relapsed/refractory MM. We seeded MM-MSCs (N=34) which had been expanded by adhesion methods in 2-D versus 3-D models in order to create an ex-vivo MM niche-like structure. In the hydrogel-based 3-D model, MM-MSCs formed compact clusters with active fibrous connections and meshwork-like structures at day 3 to 7. Moreover, calcium mineralization of clusters was observed, associated with the capacity for differentiation towards the osteoblastogenic or adipogenic lineage when cultured with differentiation media. Furthermore, the production of osteopontin (OPN) and angiopoietin-2 (Ang-2) was significantly higher in 3-D vs. 2-D MM-MSCs, assessed by multiplex luminex technology. Phenotypic profiling of 3-D MM-MSCs clusters revealed high expression of CD73+CD90+CD105+ and lack of expression of CD45, CD34 and HLA-DR, as in to 2-D MM-MSCs. MSC-specific markers including CD166 and HLA-ABC did not reveal any significant changes in 3-D vs. 2-D MM-MSCs; however, 3-D MM-MSCs had significantly decreased expression of CD271 and CD146 compared to 2-D cultures. We also observed significantly higher expression of extracellular matrix (ECM) molecules including fibronectin, laminin, collagen I, and collagen IV (p Conclusions: This 3-D co-culture system closely mimics the myeloma BM niche, and therefore may be useful to identify and validate novel targeted therapies. Disclosures No relevant conflicts of interest to declare.

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 & 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.

Extensive preclinical studies of several groups using tumor cells co-cultured with bone marrow stromal cells (BMSCs) has documented that the potent anti-MM activity of the proteasome inhibitor bortezomib is not suppressed by BMSCs (e.g. primary and immortalized BMSCs). Using our compartment-specific bioluminescence imaging (CS-BLI) assays, we extended these observations to larger panels of MM cell lines. We observed, however, a recurrent pattern that primary CD138+ MM tumor cells from bortezomib-refractory patients recurrently exhibited substantial in vitro response to clinically-achievable concentrations and durations of bortezomib treatment. To simulate this clinicopathological observation, MM.1R-Luc+ cells were injected i.v. in SCID-beige mice and treated with bortezomib (0.5 mg/kg s.c. twice weekly for 5 weeks): diffuse MM tumors initially responded to bortezomib, but eventually became refractory. These in vivo-resistant MM cells were isolated from the mice and were treated in vitro with bortezomib, exhibiteing similar responsinveness to this agent as their isogenic bortezomib-naive MM cells, To further address the possibility that this represents a previously underexplored form of a microenvironment-induced alteration in bortezomib responsiveness, we studied how MM cells respond to pharmacological proteasome inhibition after variable times of co-culture with BMSCs prior to bortezomib exposure. We observed that prolonged tumor-stromal co-culture (48-96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for many of the MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Notably, prolonged co-cultures with BMSCs prior to bortezomib treatment enhanced the activity of this agent for other MM cell lines (e.g. OPM1, Dox40, OCI-My5, KMS12BM or KMS18). However, MM.1S and MM.1R cells, when exposed to extended co-cultures with BMSCs prior to initiation of drug exposure, exhibited significant attenuation (2-3 fold increase of IC50 values) of their response to bortezomib in several independent replicate experiments. In support of these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells co-implanted with Luc-negative BMSCs did not show significant reduction in MM tumor growth with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.13), as quantified by bioluminescence imaging. In co-cultures with BMSCs, MM.1S and MM.1R cells also exhibited suppression of their response to carfilzomib (the degree of this stroma-induced resistance was more pronounced that in the case of bortezomib for these 2 cell lines). Consistent with these observations, in vivo administration of carfilzomib in the orthotopic model of diffuse bone lesions of MM.1R-Luc+ cells was associated with less pronounced reduction in tumor growth, compared to bortezomib treatment (p Disclosures: No relevant conflicts of interest to declare.

Introduction The neoplastic bone marrow (BM) milieu or so-called myeloma niche plays an essential role in initiation, development, and progression of multiple myeloma (MM). It is known that regulatory signaling molecules and cell-cell crosstalk interactions involved in the normal hematopoietic stem cell niche have also been implicated in MM. Defining phenotypic features and molecular signatures of the neoplastic BM niche in MM will provide the framework for future development of new treatment strategies targeting MM cells and their BM niche. Methods and Results To investigate the neoplastic BM microenvironment of MM patients, we compared a special gel-based 3-dimensional (3-D) model with a 2-dimensional (2-D) system for culturing MM patient-derived mesenchymal stem cells (MM-MSCs) ex-vivo. In the gel-based 3-D model, MM-MSCs formed compact clusters with active fibrous connections between clusters to create a MM niche-like structure. Phenotypic profiling of 3-D MM-MSCs clusters revealed high expression of MSC-specific markers including CD73, CD90, CD105, CD166, and HLA-ABC. On the other hand, 3-D MM-MSCs clusters lacked expression of CD45, CD34 and HLA-DR. In order to confirm differentiation potential of 3-D MM-MSC, we cultured them with differentiation media towards either osteogenic or adipogenic lineages. The lineage differentiation capacity of the 3-D system was comparable to the 2-D MM-MSCs, with stronger calcium mineralization of clusters associated with differentiation towards the osteoblastogenic lineage. Moreover, the production of osteopontin and angiopoietin-2 was evident in 3-D MM-MSCs. In these models of the neoplastic BM microenvironment in MM, significantly higher production of IL-6 (p=0.002), IL-8, MCP-1(MCAF), RANTES, VEGF and HGF (p To define the functional myeloma niche, key hematopoietic and/or neoplastic niche molecules were investigated by analyzing 30 MM-MSCs and 5 healthy volunteers MSCs. MM-MSCs cultured in 3-D vs. 2-D model have higher expression of N-cadherin and CXCL12 and decreased expression of nestin by flow cytometry analysis, reflecting the MM BM niche. Higher pattern of cytokine production of leptin, SCF and sTie-2 was evident in 3-D MM-MSCs compared to 2-D system, as well as cancer biomarkes like IGFBP-1, sEGFR and sHER2neu performed by multiplex luminex technology. Next, we observed statistically significant higher expression of extracellular matrix (ECM) molecules including fibronectin, laminin, collagen I, and collagen IV (p To investigate the pathogenesis of MM in neoplastic BM niche, we co-cultured BM aspirates of MM cells (N=30) with MM-MSCs in vitro. Co-culturing BM aspirates with (autologous) MM-MSCs increased MM cells (CD38+/CD138+/CD19-) as well as stimulated their proliferation (determined by CFSE proliferation dye) in 3-D vs. 2-D system. MM cells in 3-D MM-MSCs were 4-fold more resistant to bortezomib and pomalidomide as well as melphalan, compared to 2-D. Finally, MM cells expressing CXCR4, as well as MM side population with “stem-like” features, were significantly increased in 3-D MM-MSCs vs. 2-D MM-MSCs cultures. Conclusions Our 3-D MM-MSCs model mimics the neoplastic BM microenvironment and myeloma niche in vitro, and may be useful to identify novel targets, validate targeted therapies and determine mechanisms of drug resistance in MM. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Richardson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

Introduction A major challenge in the development of effective myeloma (MM) therapy is addressing tumor heterogeneity, with the presence of sub-clones that exhibit resistance to standard therapy. An ongoing area of investigation focuses on identification of myeloma initiating cells that demonstrate greater capacity for self-renewal and serve as a potential reservoir for disease recurrence. It has been postulated that MM cells arise from a primitive B cell precursor population distinct from the more differentiated malignant plasma cell population. The critical feature of these myeloma-propagating cells is thought to be the ability to efficiently recapitulate MM in immunocompromised mice. MUC1 is an oncoprotein aberrantly expressed in malignant cells, including multiple myeloma, that interacts with multiple transcription factors, such as NF-κB and the β-catenin/TCF4 complex, that regulate cell survival and proliferation critical for malignant transformation. We have previously demonstrated that MUC1 is expressed by AML leukemic blasts, as compared to normal hematopoietic stem cells, and blockade of MUC1 signaling prevents establishment of leukemia in immunocompromised animals. In the present project, we identify a unique population of CD34+/MUC1+/CD138+/CD20+ cells in primary MM bone marrow samples that exhibit features of myeloma initiating cells, as manifested by high levels of enzymatic ALDH activity, the ability to efflux Hoechst dye represented as “side population” (SP), and the ability to establish disease in immunocompromised mice. Of note, MM engraftment of unselected primary myeloma cells in a xenograft model has a low success rate, and typically requires the introduction of an artificial stromal support network. Methods and results Bone marrow aspirates were obtained from newly diagnosed MM patients using an established protocol approved by the IRB. Expression of MUC1, myeloid and lymphoid markers was assessed using multicolor flow cytometric analysis. While MUC1 shows only a minimal expression ( In order to study the capacity of CD34+MUC1+ cells to recapitulate MM in a murine model, a bone marrow sample was obtained from a patient with newly diagnosed MM with a cytogenetic abnormality characterized by the rearrangement of the CCND1/IGH loci. Primary bone marrow cells were fluorescently labeled and CD34-MUC+ (consistent with mature CD138+CD38hi plasma cells) and CD34+MUC+ populations of cells were isolated using FACS sorting. Cells from each population were injected into an irradiated NOD/SCID mouse (0.5x106cells/mouse). After 13 weeks, no human engraftment was detected in the 4/4 mice injected with CD34-MUC+ population of mature plasma cells. In contrast, 2/2 mice injected with CD34+MUC+ cells demonstrated human engraftment. Engrafted cells were isolated by FACS sorting and transferred onto glass slides for cytogenetic analysis by FISH. Notably, the engrafted cells harbored rearrangement at CCND1/IGH loci consistent with the originating MM clone. In another experiment, 2/2 NOD/SCID mice inoculated with CD34+MUC1+ primary MM cells demonstrated MM engraftment after 12 weeks, characterized by the presence of CD138+CD45- human plasma cells in the murine bone marrow. Conclusions We have identified a subpopulation of primitive myeloma cells that coexpress CD34 and the MUC1 oncoprotein. CD34+MUC1+ cells express CD20 and CD138, and express high levels of ALDH. CD34+MUC1+ cells demonstrate the capacity to engraft human MM cells in immunocompromised mice, even without an artificial stromal framework. Inhibition of MUC1 signaling thus may offer new avenues to target critical myeloma subclones. Disclosures: No relevant conflicts of interest to declare.

Abstract 2468 In multiple myeloma (MM) and other hematologic malignancies, bone marrow stromal cells (BMSCs) confer resistance to diverse conventional or investigational therapeutics. During the last decade, data from many groups have concurred that the in vitro anti-MM activity of the proteasome inhibitor bortezomib is very similar in the presence and absence of BMSCs, including primary and immortalized BMSCs. These well-validated observations have supported the notion that novel, more effective, therapies for the treatment of MM should ideally be, similarly to bortezomib, capable of overcoming the protective effect of BMSCs. Interestingly, however, we have observed that primary CD138+ MM tumor cells isolated from patients with clinical refractoriness to bortezomib occasionally exhibit substantial in vitro response to clinically achievable concentrations of this drug. We therefore hypothesized that, under certain previously under-explored experimental settings, BMSCs may alter the threshold of MM cell response to bortezomib-induced apoptosis. To address this hypothesis in conditions that better simulate the clinical context, we conducted compartment-specific bioluminescence imaging (CS-BLI) assays to evaluate the effect of bortezomib on tumor cells co-cultured with BMSCs for different time periods prior to bortezomib administration. We observed that prolonged tumor-stromal co-culture (48–96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for several MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Prolonged co-culture of OPM1, RPMI-8226-Dox40, OCI-My5, KMS12BM and KMS18 cells prior to bortezomib treatment enhanced its activity. Importantly, extended co-culture of MM cell lines MM.1S and MM.1R with BMSCs prior to drug treatment induced significant attenuation of their response to bortezomib, as evidenced by 2–3 fold increase of IC50 values in several independent replicate experiments and a mean % area under the bortezomib dose response curve (AUC) of 5.82% vs 14.10% in the absence vs. presence of BMSCs, respectively (p=0.0079). Consistent with these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells mixed with Luc- BMSCs did not show statistically significant reduction in MM burden with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.1320), as quantified by bioluminescence imaging. In contrast, the same dose and schedule of bortezomib treatment significantly suppressed tumor burden, compared to vehicle-treated controls, of monotypic s.c. xenografts of Luc+ MM.1S cells in SCID mice (p=0.0022), as in prior experience. To evaluate the molecular mechanisms of cell non-autonomous decrease in MM cell response to bortezomib, we compared the transcriptional profiles of MM.1S cells in extended co-cultures with HS-5 BMSCs vs. MM.1S cells cultured in isolation. These studies identified a distinct transcriptional signature of stroma-induced transcripts, including several (e.g. PSMC3, ITGB7, FOS, ALDH1L2) for which transcript expression higher than the median levels for refractory MM patients correlated with shorter overall survival (p Disclosures: Groen: Genmab BV: Research Funding. McMilllin:Axios Biosciences: Equity Ownership. Mitsiades:Millennium Pharmaceuticals: Honoraria; Celgene: Honoraria; Novartis Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Merck &Co.: Honoraria; Centocor: Honoraria; Arno Therapeutics: Honoraria; Amgen: Research Funding; AVEO Pharma: Research Funding; OSI: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Johnson & Johnson: Research Funding; PharmaMar: Licensing royalties Other; Axios Biosciences: Uncompensated Role as advisor, Uncompensated Role as advisor Other.

Abstract 1812 The neoplastic bone marrow (BM) milieu plays important roles in the development of multiple myeloma (MM). Specifically, cell-cell crosstalk interactions and regulatory signaling molecules involved in the normal hematopoietic stem cell niche have also been implicated in initiation, development, and progression of MM. Moreover, this “myeloma niche” serves as a sanctuary site for MM stem cells. The increasing knowledge of interactions between complex of MM cells and other normal hematopoietic cells and also non-hematopoietic mesenchymal stem cells and their progeny may enhance our understanding of MM cell biology and pathogenesis as well as provide the basis for novel therapeutic strategies. In order to develop an experimental system mimicking the specialized properties of the neoplastic BM microenvironment, we used hydrogel based 3-dimensional (3-D) versus 2-dimensional (2-D) models together with MM-derived mesenchymal stem cells (MM-MSCs) to create MM niche-like structures in vitro. In this 3-D model, MM-MSCs grew with the matrix fibers and at day 7 formed stable compact clusters, with fibrous meshwork-like structure among them. Furthermore, we showed tri-lineage differentiation capacity of 3-D comparable to 2D MM-MSCs towards osteogenic, adipogenic, and chondrogenic lineages. Our 3-D MM-MSCs model also showed calcium mineralization of clusters associated with differentiation towards the osteoblastogenic lineage. The phenotypic profile of MM-MSCs grown in the 2-D vs. 3-D model using MSC-specific markers (such as CD166, HLA-ABC, CD73, CD105, and CD90) did not reveal any significant changes, however CD271 was more highly expressed in the 3-D model, while CD146 was overexpressed in 2-D cultures. Because the stem cell niche is enriched with extracellular matrix (ECM) molecules resulting in an overexpansion of the stem cell pool, we studied whether expression of these molecules was conserved in the 3-D model. Analyzing 30 MM-MSCs vs. 5 normal donor MSCs, we observed that the 3-D MM-MSCs expressed significantly high level of ECM molecules including laminin, fibronectin, collagen I, collagen IV and vitronectin (p To mimic the functional marrow niche of MM in our model, we cultured the tumor cells from MM patients (N=30) with their autologous MM-MSCs in vitro. MM plasma cells (identified as CD38/CD138+ cells) were increased in percentage and proliferation after 14 days in the 3-D vs. 2-D model. Finally, MM cells expressing CXCR4, as well as MM side population with “stem-like” features (Jakubikova J et al., Blood 2011) were also significantly increased in 3-D MM-MSCs vs. 2-D MM-MSCs cultures. Our study shows that hydrogel based 3-D MM-MSCs model forms a functional niche in vitro, and may be useful to identify novel targets and validate therapies directed at MM stem cells in the MM BM niche. Disclosures: Munshi: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; Oncopep: Patents & Royalties. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene Corp: Membership on an entity's Board of Directors or advisory committees.

Abstract 652 Long-term survival of patients with acute myeloid leukemia (AML) is poor, and new forms of therapy are needed. Many genetic lesions have been identified and studied, and most patients have chromosome translocations or other mutations that promote self-renewal of leukemic stem cells, block differentiation, enhance growth, and block apoptosis. Only a few of these mutations result in druggable targets (e.g., PML-RARa, Kit, PDGFR, FLT3 for instance). In addition to genetic lesions, epigenetic abnormalities have been shown to be very common in AML, and provide opportunities for novel treatments. Using genome-wide approaches to identify alternative splicing, we have recently shown that AML cells have a high level of aberrantly regulated genome-wide alternative splicing (AS) as a frequent epigenetic event. By comparing samples from 62 AML patients with 10 normal donors (NDs) we identified 428 genes differentially spliced in AML. A list of differentially spliced genes includes 50 oncogenes and 52 tumor suppressor genes, as well as genes encoding proteins involved in cell proliferation and differentiation, and apoptosis. We evaluated splicing event frequency in AML compared to NDs and we observed that on average 527 (range 137–1657) genes were identified as differentially spliced in any given patient, out of 62 analyzed. Also, we found that any given differentially spliced gene, of the 3,108 detected, were spliced on average in 26 (range 1–54) AML patients. Thus, splicing aberrations are highly recurrent in AML patients. To identify the causes of aberrant splicing in AML, we evaluated transcript levels of the 24 major splicing factors (SFs) that are involved in the first and second splicing transesterification reactions. These splicing factors are important proteins involved in spliceosomalassembly. Expression levels of these SFs were evaluated in 20 AML patients exhibiting high levels of AS. Quantitative RT-PCR analysis showed significant (up to 30 fold) upregulation of U2AF2 (P We have obtained a minigene cassette of the p53 inducible PIG3 gene based on previous splicing studies. The minigene cassette was cloned between RFP (red fluorescent prtoein) and GFP (green fluorescent prtoein) in such a way that translation of the normally spliced transcript results in expression of RFP and GFP, while aberrant splicing results in the expression of RFP only. Production of a similar minigene cassette that includes exons/introns of a gene that is subjected to aberrant splicing in AML (NOTCH2, FLT3 and CD13) is in progress. In studies, completed so far, with the PIG3 minigene cassette construct transiently transfected into the HEK293 cells lines, overexpression of PTBP increased aberrant splicing of the PIG3 minigene. Similar studies testing the effects of elevated levels of U2AF2 and PTBP on NOTCH2 and other genes misspliced in AML (such as FLT3 and CD13) will be presented. Our results indicate that aberrant splicing could be an important event in AML, and development of an in vitro, synthetic splicing assay will enable us to better understand the underlying causes of this process in AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 1838 Introduction: Graft-versus-myeloma (GvM) effect following allogeneic stem cell transplantation (allo-SCT) is well established and often accompanied by graft-versus-host disease (GVHD). Unfortunately, most patients still relapse following allo-SCT, underscoring the need for new transplant strategies. Interestingly, studies evaluating the proteasome inhibitor bortezomib combined with donor lymphocyte infusions (DLI) in myeloma patients suggest enhanced efficacy of the combination. In contrast, other reports demonstrate that bortezomib has activity in preventing GVHD in various malignancies, including myeloma. Here, we investigate the effects of bortezomib on T cell-mediated myeloma cell killing in vitro. Methods: Tumor cell compartment-specific bioluminescence imaging (CS-BLI) technology was used to monitor the cytotoxic effects of T cells on myeloma cells in the presence or absence of antimyeloma agents. To this end, myeloma cell lines were stably transduced with a firefly luciferase construct. Two different T cell clones were used: 1) the alloreactive CD8+-T cell clone allo-A2, which kills all HLA-A2-positive cells, including the myeloma cell line U266; and 2) the CD4+ T-cell clone 3AB11, generated from a myeloma patient with clinical GvM/GVHD after allo-SCT. 3AB11 cells recognize a hematopoietically restricted minor antigen, also presented by the myeloma cell line UM9. Protein expression was determined by western blot analysis. Results: The CD4+-T cell clone 3AB11 and the allo-A2 specific CD8+- T cell clone displayed a time- and dose-dependent lysis of myeloma cell lines UM9 and U266, respectively. Concurrent treatment of myeloma cells with the T cell clones and bortezomib resulted in synergistic enhancement of myeloma cell death, compared to T cells alone or bortezomib alone. This synergism was observed at 24, 48, and 72 hours (but not after 4 hours) of co-treatment. Dexamethasone, which significantly inhibited myeloma cell death induced by T cells, functioned as control. When myeloma cells were treated for 12 hours with bortezomib and then extensively washed, there was still enhancement of T cell-mediated killing. However, pre-incubation of T cells with bortezomib significantly impaired their ability to induce myeloma cell lysis. These washout experiments indicate that bortezomib sensitizes myeloma cells to T cell-mediated killing, but does not enhance the intrinsic cytotoxic activity of T cells. Mechanistic studies demonstrated that antibodies blocking FAS ligand partially impaired myeloma cell lysis by 3AB11 and allo-A2 clones. More interestingly, however, blockade of FAS/FAS ligand interactions completely abrogated the synergism between bortezomib and T cells. Antibodies against TRAIL had no such effects. Furthermore, western blot analyses revealed that in UM9 and U266 cells bortezomib reduced protein levels of FLICE inhibitory protein (cFLIP), which is a potent inhibitor of FAS ligand-induced apoptosis. These results support the idea that bortezomib enhances the sensitivity of myeloma cells toward T cell-mediated lysis by restoring apoptosis signaling via the FAS/FAS ligand pathway. Conclusion: Proteasome inhibition sensitizes myeloma cells to T cell-mediated killing via a FAS/FAS ligand-dependent mechanism. Conversely, bortezomib impairs T cell function. However, the net effect is a synergistic killing of myeloma cells when T cells and bortezomib are combined. These data support further exploration of the use of bortezomib in preclinical models, with derived clinical trials in the allogeneic setting (e.g. in combination with DLI or following allo-SCT) or in the autologous setting (e.g. in combination with a tumor vaccine) potentially warranted. (CSM and TM are joint senior authors) Disclosures: van de Donk: Celgene: Research Funding. Lokhorst:Celgene: Research Funding; Jansen-Cilag: Research Funding. Hayes:Pfizer: Research Funding; Amgen: Research Funding. Munshi:Celgene: Consultancy; Millenium: Consultancy; Novartis: Consultancy; Onyx: Consultancy. Richardson:Millennium:; Celgene:; Johnson & Johnson:; Novartis:; Bristol Myers Squibb:. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Amnis Therapeutics: Consultancy, Honoraria; PharmaMar: Licencing royalties; OSI Pharmaceuticals: Research Funding; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding.

Abstract 2913 CRM1 (chromosomal region maintenance 1, XPO1) is the major export protein regulating degradation of key tumor suppressors by transporting them from nucleus to cytoplasm, thus abrogating their function. Highly expressed CRM1 is associated with poor prognosis in several solid tumors, and inhibition of CRM1 restores function of tumor suppressors such as p53, p21 and FOXO and IκB (cellular antagonist of NF-κB). Furthermore, CRM1 knockdown enhances sensitivity of human multiple myeloma (MM) cell lines to topoisomerase II inhibitor (Cancer Res 2009 :69, 17). In this study, we investigated a novel selective CRM1 inhibitor, KPT-185, in human MM cells. Gene expression profiling analysis using GEO database showed that CRM1 expression is significantly increased in CD138+ cells from MM patients versus monoclonal gammopathy of undetermined significance (MGUS) patients or normal donors (p Disclosures: Landesman: Karyopharm Therapeutics: Employment. Senapedis:Karyopharm Therapeutics: Employment. Shacham:Karyopharm Therapeutics Inc.: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. McCauley:Karyopharm Therapeutic Inc.: Employment. Saint-Martin:Karyopharm Therapeutics Inc: Employment. Munshi:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.

Abstract 1820 Proteasome inhibitors such as Bortezomib (Bort) represent a key drug class for the therapeutic management of multiple myeloma (MM). However, MM patients, even those who initially achieve complete clinical and biochemical remission with bortezomib-based regimens eventually relapse, and bortezomib-refractory disease is associated with short overall survival. Identifying the molecular basis of resistance to bortezomib is therefore crucial for the rational development of novel therapies to hopefully improve clinical outcome for advanced MM. To address this question, we generated an isogenic cell line model of bortezomib resistance by successive rounds of in vitro exposure of bortezomib-sensitive MM.1R cells to progressively increasing bortezomib concentrations. Serial dose-response analyses confirmed the generation of several clones with variable reduction in bortezomib-sensitivity (IC50 range 40–80nM vs. Disclosures: Hayes: Pfizer: Research Funding; Amgen: Research Funding. van de Donk:Celgene: Research Funding. Richardson:Johnson & Johnson: Advisory Board; Millennium: Advisory Board; Celgene: Advisory Board; Bristol-Myers Squibb: Advisory Board; Novartis: Advisory Board. Anderson:Millennium: Consultancy, Research Funding. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Amnis Therapeutics: Consultancy, Honoraria; PharmaMar.: Licensinig royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding.

Abstract 5016 Introduction: Multiple myeloma (MM) is a hematologic malignancy characterized by an abundant clonal proliferation of plasma cells in the bone marrow. Despite significant recent progress in the treatment of MM, this plasma cell dyscrasia remains incurable. Therefore, the pursuit of new investigational therapeutic agents, used alone or in combination is critically important to improve patient outcome. In our previous studies, we introduced plant isothiocyanates (ITCs) as compounds with potent cytotoxicity against MM cell lines and primary MM samples. The effect of these compounds was associated with pleitotropic effects on signal transduction pathways regulating the apoptotic threshold of MM cells. This led us to hypothesize that ITCs could be rationally combined with other anti-MM agents and enhance their anti-tumor activity. Methods and Results: On the basis of previous studies in different cancer types, we examined whether the ITCs, suforaphane (SFN) and PEITC, showed inhibitory activity against histone deacetylases (HDAC) in MM. We found that ITCs, SFN and PEITC showed inhibitory activity against histone deacetylases (HDAC) using fluorometric HDAC assay. Therefore, we tested combination treatment of ITCs with the HDAC inhibitor vorinostat. This combination had potent synergistic effect against MM cells, and was associated with apoptotic cell death following G2/M cell cycle block and depletion of mitochondrial potential. Interestingly, synergistic inhibition of HDACs was not observed. In addition, combination had additive and synergistic effect on phosphorylation of c-Jun and p38MAPK, respectively. Because the BM microenvironment is considered a key regulator of MM cell biological behavior, we evaluated the impact of bone marrow stromal cells (BMSCs) on the combination treatment. Our co-culture system where MM cells were labeled with CFSE proliferation dye, and therefore easily distinguished from BMSCs, the interaction between these cell populations did not confer MM cells resistance to the combination treatment. Next, we evaluated combinations of ITCs with established anti-MM agents. We observed that combination of ITCs with melphalan and dexamethasone led to synergistic induction of MM cells apoptosis, with activation of caspases -8 and -9, and cleavage of PARP. The synergistic effect of the ITC combination with melphalan was not abrogated by the MM cell co-culture with stroma, while dexamethasone synergized only with SFN. The combination of SFN and melphalan synergistically increased activation of p53 and p38MAPK at later time point than PEITC and melphalan. Dexamethasone alone activated NFkB and p38MAPK, but combination with ITCs decreased the phosphorylation of both signaling molecules. Conclusions: Our study raises the intriguing possibility that dietary supplementation with ITCs during the treatment of MM patients could potentially modulate therapeutic responses. Our results specifically indicate that ITCs can enhance the activity of certain conventional and novel anti-MM agents, providing the preclinical framework for future clinical studies of combination regimens that include ITCs for the treatment of MM. Disclosures: Richardson: Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Camp;o.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Abstract 1574 Cancer cells with stem cell-like features are a topic of intense research because their resistance to existing drugs is considered a culprit for relapses, even in patients with complete remission defined by clinical, biochemical and imaging parameters or by sensitive molecular techniques. Salinomycin, an antibacterial and coccidiodostatic ionophore, is reported (Cell 2009;138(4):645-59) to be >100-fold more potent against breast cancer cells with stem cell-like phenotype after mesenchymal transdifferentiation due to stable transfection with shRNA against CDH1 than against the parental cells. We evaluated whether salinomycin could also exhibit a similar activity against stem cell-like cells in multiple myeloma (MM). To establish a comparative reference for such potential activity, we first tested salinomycin (0-10 uM for up to 72hrs) against a panel of 15 MM cell lines and observed IC50 values 80% reduction of tumor cell viability in 6/15 cell lines tested at 0.5 uM, i.e. levels lower than the IC50 values for in vitro activity of salinomycin against breast cancer cells with (HMLE-shCDH1, IC50 ∼1 uM) or without (HMLE-shControl, IC50 >>10 uM) stem cell-like features. CD138+ purified primary tumor cells from 3 MM patients responded to salinomycin with IC50 values (105, 332 and 750 nM, respectively) in the same range as MM cell lines. In vitro combinations with bortezomib, doxorubicin, melphalan, and dexamethasone showed overall no antagonism, while evidence of additive or even synergistic effect could be identified in certain dose ranges. Because MM cell lines and primary tumor cells responded concordantly to salinomycin and with higher sensitivity than breast cancer stem cell-like cells, we hypothesized that MM cells may in general be more responsive to salinomycin than other tumors. Since tumor-stromal interactions can increase the expression of transcriptional signatures of “stemness” in MM cells, we embarked on characterizing the anti-MM properties of salinomycin using compartment-specific bioluminescence imaging (CSBLI) assays. These showed that co-culture with stromal cells did not confer resistance to salinomycin in 5 MM cell lines (MM.1S, OCI-My5, KMS-11, KMS-18, NCI-H929) and in fact enhanced its activity against 4 of them. Side population (SP) cells, defined by their ability to efflux Hoechst stain, represent a stem cell-like population which was identified in MM cell lines and could represent the functional equivalent of the mesenchymally transdifferentiated breast cancer stem cell-like cells. We observed that salinomycin reduces the SP fraction of MM cell lines at doses >20 times lower than those required for in vitro effect against the bulk <> of the respective cell lines. Interestingly, the anti-SP effect of salinomycin was more pronounced in the presence of stroma, similarly to the CSBLI studies on the entire MM cell population and consistent with our prior observation that tumor-stroma interaction enhances transcriptional signatures of ≪stemness≫ in the tumor compartment. However, when we tested the in vivo anti-MM activity of salinomycin in an orthotopic model of i.v. injected Luc+ MM cells, no anti-MM activity (in terms of tumor burden decrease or overall survival prolongation) was observed at the maximum tolerated dose (1 mg/kg i.p. daily, which is consistent with most studies reported thus far in the literature). Ex vivo treatment of KMS-11 cells with salinomycin doses (100 nM for 72 hrs) selectively targeting SP cells was followed by s.c. injection of these cells or vehicle-treated controls in sublethallly irradiated SCID/NOD mice, but no statistically significant improvement in tumor burden or overall survival was observed. Our in vitro results indicate that salinomycin exhibits intriguing in vitro anti-MM activity, not only against SP cells but also against the bulk ≪main≫ MM cell population, even in the presence of stromal support. In contrast, the in vivo activity of salinomycin is compromised by side effects in the orthotopic model of MM lesions, while short term ex vivo exposure of tumor cells is conceivably insufficient to eradicate clonogenic cells and lead to appreciable delay in tumor growth in vivo. Our studies point to intriguing features as well as notable challenges that have to overcome before salinomycin or other more selective agents of this class can be safely tested in clinical trials in MM. Disclosures: McMillin: Axios Biosciences: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Abstract 1581 Introduction: Recurrence of multiple myeloma (MM) after establishment of complete remission indicates the presence of a small fraction of resistant tumor cells which persists after treatment and expresses stem cell-like characteristics. In our previous studies, we confirmed the existence of a MM population with “stem-like” features, known as side population (SP) cells. SP cells exhibit substantial heterogeneity not only in MM cell lines, but also in primary samples. Importantly, SP cells manifest significant clonogenic potential in vitro compared to the main population (MP). In our current study, we examined whether the clonogenicity of SP cells in vitro is associated with tumorigenic potential of SP cells in vivo. Methods and Results: To evaluate tumorigenicity of SP cells, several dilutions of sorted SP and MP cells were injected into immunocompromised mice. In CB17/SCID mice, sorted SP cells (range 105 - 106 cells/mouse) from OPM1 and KMS11 cell lines induced significantly greater tumor growth compared to MP cells (90-70% less tumor mass than SP tumors). At lower innocula of cells (10 × 103 cells/mouse), SP cells resulted in tumors, whereas no tumors were produced by MP cells. In NOD/SCID mice, lower numbers of SP cells (1 × 103 cells/mouse) produced tumor. These data strongly support the tumorigenic potential of SP sub-population and provide the rationale for targeting these cells in novel therapeutic strategies in MM. Indeed, our previous studies show that lenalidomide significantly decreased the percentage and clonogenicity of SP cells. We therefore expanded these studies to next examine whether other novel agents target SP cells. Novel agents pomalidomide (1 μM; decreased by 97%) and histone deacetylase (HDAC) inhibitor vorinostat (0.4 μM; decreased by 86%) significantly affected SP cells. Natural plant compound sulforaphane (2 μM; decreased by 86%), but not isothiocyanate PEITC, also decreased percentage of SP cells. We similarly evaluated the anti-SP effect of compounds known or proposed to target crucial signaling pathways in hematopoietic/cancer stem cells including cantharidin (terpenoid inhibitor of PPA2) and its demethylated analog norcantharin, deguelin (PI3K/Akt inhibitor), plumbagin (quinonoid substrate of ABCG2) and ionophore salinomycin. All these compounds significantly decreased or completely eliminated SP cells at low nontoxic doses in flow cytometry-based Hoechst 33342 staining assays. However, clonogenicity of SP cells treated by salinomycin was only slightly affected compared to control SP cells by clonogenic assay in vitro. In addition, Hedgehog pathway inhibitor cyclopamine (10 μM; decreased by 82%), as well as its analog tomatidine (10 μM; decreased by 64%), significantly decreased the percentage of SP cells, while sonic hedgehog agonist purmorphamine (0.8 μM; decreased by 17%) only modestly decreased the percentage of SP cells. To study these substantial inhibitory effects of tested compounds, the clonogenic assays were performed in vitro. Finally, the effect of drugs and compounds against SP cells was also evaluated in co-culture with bone marrow stromal cells, which confer growth and survival in MM. Importantly, the SP fraction was decreased by these agents, whereas no effect of hedgehog agonist and plumbagin was detected. Conclusions: Our studies confirmed the tumorigenic potential of SP cells isolated from established MM cell lines and identify the impact of targeted therapies against these cells, providing the rationale for further studies to evaluate the clinical potential of therapeutic targeting of SP cells in MM. Disclosures: Richardson: Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Abstract 730 Multi-targeted kinase inhibitors, when associated with manageable toxicity, offer the therapeutically desirable option of targeting, through a single chemical entity, several pathways that may contribute to the complexity and heterogeneity of molecular lesions harbored by neoplasias such as multiple myeloma (MM). However, intractable questions often emerge while prioritizing for preclinical studies different multi-targeted agents with extensive and/or only partially overlapping of sets of known targets. We have hypothesized that the potential therapeutic relevance of a multi-targeted inhibitor may be reflected on the prognostic relevance of its targets' transcriptional signature. We applied this concept in the case of the orally bioavailable multi-targeted kinase inhibitor AS703569, which targets (with IC50 in low nM range) all 3 Aurora kinase (AK) isoforms as well as various other kinases (e.g. cSRC, FGFR1, Flt3, Fyn, Lyn, Rsk1-3, Yes, Axl, et.c.) and evaluated the transcriptional signature of AS703569 kinase targets (with IC50 Disclosures: Laubach: Novartis: Consultancy, Honoraria. Rastelli:EMD Serono: Employment. Clark:EMD Serono: Employment. Sarno:EMD Serono: Employment. Richardson:Millenium: (Speakers' Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers' Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis : Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck & Co: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono : Research Funding; Sunesis Pharmaceuticals: Research Funding.

Abstract 1729 Poster Board I-755 Background Multidrug transporters are energy-dependent transmembrane proteins which can efflux a broad range of anticancer drugs and thereby play a role in resistance to the actions of substrate agents. Classically, three transporters, p-glycoprotein (Pgp; MDR-1; ABCB1), multidrug resistant protein-1 (MRP-1; ABCC1) and breast cancer resistance protein (BCRP; MXR; ABCG2), have been found to have the broadest substrate specificity and a strong correlation with drug resistance in vitro and in vivo in many models and forms of cancer. We have sought to characterize the interaction of bortezomib with these transporters and thereby explore the potential for these agents to play a role in resistance. Bortezomib is a novel proteosome inhibitor with significant activity in multiple myeloma, although subsets of patients remain refractory to the activity of the drug. Hence, better characterization of the interactions of this drug with classical resistance mechanisms may identify improved treatment applications. Methods and Results We investigated the role of these transporters by using isogenic cell line models which are resistant due to overexpression of a particular transporter: DLKP lung cancer cell line that overexpresses MRP-1; DLKP-A which overexpresses Pgp; and DLKP-SQ-Mitox which overexpresses BCRP. DLKP-A cells exhibited a 4.6-fold decrease in responsiveness to bortezomib compared to parental DLKP cells. In DLKP-SQ-Mitox, bortezomib-induced cytotoxicity was comparable to DLKP. When bortezomib was combined with elacridar, a Pgp and BCRP inhibitor, significant synergy was evident in DLKP-A (100% viable cells with single agent treatment versus 11% with the combination), but not DLKP-SQ-Mitox. Sulindac, an MRP-1 inhibitor, combined with bortezomib failed to produce any synergy in MRP-1 positive DLKP cells. Conversely, combination assays of Pgp substrate cytotoxics such as doxorubicin with Bortezomib were largely additive in nature. This indicates that bortezomib has little, if any, direct Pgp inhibitory activity, as combinations of a traditional Pgp inhibitor (such as elacridar) and doxorubicin would show marked synergy rather than just an additive effect in Pgp positive cells. To further characterize the extent of this interaction with Pgp, we conducted cytotoxicity assays in cell lines with varying levels of Pgp overexpression. NCI/Adr-res (ovarian cancer, high Pgp overexpression), RPMI-Dox40 (multiple myeloma, moderate Pgp overexpression) and A549-taxol (lung cancer, low Pgp overexpression). The combination of bortezomib and elacridar that produced the most synergy was in cell lines expressing moderate to high levels of Pgp expression. Cell lines with lower Pgp expression produced an additive cytotoxicity. We next examined whether bortezomib had any direct effect on Pgp expression. In RPMI-Dox40 cells, Pgp expression is reduced in a time-dependent manner with bortezomib treatment. Conclusions Our studies therefore show that bortezomib is a substrate for Pgp but not the other drug efflux pumps. In tumor cells expressing high levels of Pgp, the efficacy of bortezomib is synergistically enhanced by combinations with a Pgp inhibitor, while bortezomib treatment itself can reduce the expression of Pgp. This study suggests that in the subset of patients with advanced multiple myeloma or solid tumors which express high levels of Pgp, inhibition of its function could contribute to enhanced responsiveness to bortezomib. Disclosures Richardson: millenium: Membership on an entity's Board of Directors or advisory committees, Research Funding; celgene: Membership on an entity's Board of Directors or advisory committees, speakers bureau up to 7/1/09; MLNM: speakers bureau up to 7/1/09. Mitsiades:Millennium Pharmaceuticals : Consultancy, Honoraria; Novartis Pharmaceuticals : Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: licensing royalties ; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono : Research Funding; Sunesis Pharmaceuticals: Research Funding. Anderson:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Biotest AG: Consultancy, Research Funding.

Abstract 954 Introduction: Multiple myeloma (MM), a malignancy of plasma cells, remains incurable even though newly established regimens incorporating conventional and novel agents are capable of achieving complete clinical and biochemical remissions in a sizeable subset of MM patients. This indicates the presence of a distinctive drug-resistant sub-population of MM cells, which might be responsible for tumor re-growth. Methods and Results: To evaluate this phenomenon, we performed flow cytometry-based Hoechst 33342 staining assay to evaluate the existence and characteristics in MM of a population known as side population (SP), which has been identified in diverse neoplasias and has been reported to exhibit “stem-like” features. We specifically performed flow cytometry or ImageStream analyses to quantify the SP population in a panel of 18 MM cell lines. These analyses revealed heterogeneity in the proportion of SP fractions in different cell lines and indicated the lack of correlation between CD138 expression and SP fraction. We observed that, after sorting of the SP and non-SP fractions, SP cells have the potential to generate colonies in CFU assays, and regenerate a population resembling the original, pre-sorted, one. Moreover, SP cells had a high proliferation index compared to non-SP cells. We also observed that most MM cells lines express significantly higher levels of ABCG2 transcripts in their sorted SP fraction compared to their non-SP cells. The mRNA profile results were further corroborated by functional assays, where SP cells showed more pronounced efflux of the known ABCG2 substrate mitoxantrone, consistent with the association of SP phenotype with ABCG2 expression. Interestingly, at concentrations and durations of drug exposure that did not substantially affect the viability of non-SP cells, the immunomodulatory thalidomide derivative (IMiDs) lenalidomide significantly decreased the percentage and clonogenicity of SP cells. This response was accompanied by a distinct pattern of changes in diverse signaling pathways in SP cells (including phosphorylation changes in Akt, GSK-3a/b, MEK1, c-Jun, p53, p38 MAPK, p70S6K and STAT3) and increase of CD138-/low+ phenotype. Because the BM microenvironment is considered a key regulator of MM cell biological behavior, we evaluated the impact of bone marrow stromal cells (BMSCs) on the behavior of SP cells and observed that BMSCs led to increased percentage, viability, as well as proliferation potential of SP cells. Interestingly, IMiDs abrogated this stimulatory effect of stromal cells by significantly decreasing SP cell percentages. In these co-culture models, we identified several cytokines, including IL-1b, IL-10, IL-17; chemokines (MIP-1a, MIP-1b and IP-10) and growth factors such as GM-CSF and VEGF, which were up-regulated in co-culture MM cells with stromal cells compared to stroma alone or MM cells alone. Significant modulation in the production of these cytokines and chemokines was detected after IMiD treatment of MM cells only, as well as in co-culture model. Conclusion: Identifying the clonogenic population of SP cells could facilitate the development of new strategies targeting subpopulations of MM cells with drug resistant properties. Our study raises the intriguing possibility that IMiDs could represent such a strategy that can target SP cells and counteract their resistance to different conventional, and perhaps some novel, therapies. In addition, our data suggest that further studies of this tumor cell population are warranted towards to the goal of developing new strategies to treat MM. Disclosures: Laubach: Novartis: Consultancy, Honoraria. Richardson:Millenium: (Speakers' bureau until 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers' bureau until 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis : Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono : Research Funding; Sunesis Pharmaceuticals: Research Funding.

Abstract 1741 Poster Board I-767 Background In contrast to most normal cells, cancer cells typically produce energy predominantly by glycolysis as demonstrated by O. Warburg more than 50 years ago. Methyljasmonate (MJ), a hormone produced by plants in response to biotic & abiotic stresses such as herbivory and wounding, has been shown to prevent the interaction of hexokinase (Hxk) and voltage dependent anion channels (VDACs), thereby significantly impacting the onset of glycolytic energy production. This may explain promising preclinical results observed with MJ against a variety of cancer cells, including myeloid leukemia and B-cell lymphoma cell lines. Methods and Results We tested the potential of MJ against Multiple Myeloma (MM) cells. We first evaluated the response of 16 different MM cell lines to 24 h of exposure to MJ concentrations of 0.5 – 3.5 mM using MTT assays. 15/16 of the MM cell lines tested displayed an IC50 of < 1.5 mM. In contrast, HS-5 stroma cells and peripheral blood mononuclear cells (PBMCs) did not respond to that MJ concentration, and even at a concentration of 2.5 mM MJ showed a maximal reduction of cell viability of 40%. Similarly to MM cell lines, purified CD138+ primary tumor cells of 3 MM patients displayed an IC50 of < 1.5 mM, suggesting that the differential sensitivity of MM vs. normal cells to MJ is not restricted to cell lines, but is also observed with primary tumor cells. Importantly, neither co-culture with HS-5 stroma nor IL-6 protected MM cells against MJ. Cell death commitment assays revealed that 1h exposure of 1.5 mM MJ induced cell death. Annexin V/PI FACS analysis of MJ-exposed MM cells showed that the cell death is mainly driven by apoptosis, evidenced by cleavage of caspases 3, 8 and 9 as well as of PARP. However, pre-incubation of MM cells with specific caspase inhibitors such as 10 mM of AC-DEVD-CHO, Z-IETD-fmk, Z-LEHD-fmk or 50 mM of Z-VAD only minimally protects the cancer cells from MJ exposure. Therefore, the impact of the MJ is not solely due to caspase triggered proteolytic cascades. Measurements of cellular ATP content by cell titer glow (CTG; Promega, Madison, WI) assay showed rapid depletion of ATP triggered by MJ action in sensitive MM cell lines. Additionally, we observed that 1 h exposure to 2 mM MJ modulated signaling pathways including IRS1/PI3K/AKT, MEK1/2, as well as Stat3 and JNK. FACS-based cell cycle analysis after propidium iodide staining did not show cell cycle arrest, but rather a rapid transition of cells to G0/G1 No correlation of sensitivity of MM cell lines and the number of mitochondria per cancer cell, as determined by Mitotracker Green (Invitrogen, Carlsbad, CA) -based flow analysis, was observed. We next examined if MJ exhibits either significant antagonism or synergy with established or novel anti-MM agents, including Bortezomib, Lenalidomide, Doxorubicin, Rapamycin or Dexamethasone, but discovered neither. However, MJ displayed synergy when combined with 2-Deoxyglucose. Finally, MJ was tested in vivo in scid/nod mice irradiated with 150 rads, injected with 1× 106 MM1S cells, and then, treated at 500 mg/kg by IP administration on a 5 days on / 2 days off schedule starting two weeks after tumor cell injection, There was an overall survival advantage of MJ-treated animals over the respective controls, with all treated mice (n=10) still alive but 6/10 control mice dead after 27 d. Conclusions Based on its rapidity of anti-MM action, favorable safety profile in preclinical models, distinct pattern of molecular sequelae, and compatibility with established anti-MM agents, MJ represents a promising investigational anti-MM agent. Disclosures Laubach: Novartis: Consultancy, Honoraria. Richardson:Millennium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

Context: Novel therapeutic strategies targeting cell cycle regulation are attractive for multiple myeloma (MM) because of the increased proliferative index in advanced drug-resistant disease. Having previously studied the role of Cdk inhibition in MM, we have looked at the cell cycle-related polo kinases (PLKs), because their expression is associated with adverse prognosis in solid tumors. We report preclinical studies on the anti-MM activity of the PLK1/2/3 small molecule inhibitor BI2536. Methods/Results: We tested 39 human tumor cell lines by MTT colormetric assay, including MM (n=26), T-ALL (n=8), solid tumors (n=5), as well normal human tissues. BI2536 exhibited activity in the low nano-molar range with IC50 values Conclusion: Proteins pivotal for cell cycle progression represent promising targets for treating highly proliferating tumors. Treatment of MM with a PLK inhibitor provides evidence that polo kinases are promising targets for MM therapy. Importantly, BI2536 activity was enhanced in the presence of stromal cells, providing evidence that this class of compounds will be active in the tumor microenvironment.

Isothiocyanates (ITCs) represent a family of phytochemicals found in cruciferous vegetables. Several epidemiological studies indicated that high intake of diet-derived ITCs may provide chemopreventive effect associated with a reduced risk of renal, prostate, pancreatic and colorectal carcinoma. ITCs have also been reported to enhance the chemosensitivity of diverse types of tumor cells. We therefore evaluated the cytotoxic effect of ITC, sulforaphane (SFN) and phenylethyl isothiocyanate (PEITC) on a panel of human MM cell lines, including cells resistant to doxorubicin (RPMI-Dox40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5), and dexamethasone (MM.1R, OPM1 and OPM2); as well as cells with low sensitivity to thalidomide derivatives (RPMI 8226-S) and sensitive cell lines (MM.1S). We evaluated the anti-MM activity of these compounds using both MTT and flow cytometric assays. Our results suggest that all tested MM cell lines are susceptible to the cytotoxic effect of both ITCs at concentrations in the same order of magnitude as those achieved in vivo by dietary consumption of cruciferous vegetables. PEITC (IC50 of 3.5–8.2 μM) was more potent than SFN (IC50 of 5–15 mM) at 48 h. ITCs induce apoptotic death of MM cells, evidenced by Annexin V-FITC staining, increased sub-G1 population measured by flow cytometry, and cleavage of PARP and caspase-3 by western blot analysis, ITCs also induced G2/M cell cycle arrest and depletion of mitochondrial potential in JC-1 probed MM cell lines. Multiplex analysis of phoshorylation signaling pathways, using the Luminex system and confirmed by conventional western blot analysis, revealed that PEITC at 2hrs triggers MAPK activation (MEK1 (4-fold), ERK1/2 (4.4-fold), JNK (25.4-fold) and p38MAPK (6-fold)) at 2h, which likely are stress responses of cells to PEITC treatment. SFN induced less pronounced but a more sustained MAPK activation (up to 24 h) than PEITC. Concentration-dependent increases in phosphorylation of GSK3α/β, c-jun, p70S6 kinase, and p90RSK were also observed at early time-points after ITCs-treatment. In contrast, decreased phoshorylation of Akt was observed in MM cells treated with SFN at 2 h and PEITC at 24 h. Chou-Talalay analysis of the effects of combinations of ITCs with anti-MM drugs (dexamethasone, melphalan and bortezomib) revealed all combinations to have synergistic MM cytotoxicity. Importantly, ITCs treatment, both alone and in combination with the aforementioned agents, also significantly suppressed proliferation of CFSE-labeled MM cell lines co-cultured with the human bone stromal cell line HS-5. These results indicate that SFN and PEITC suppress survival and proliferation of MM cells, both alone and in combination, and suggest their therapeutic potential in MM.

Halofuginone, a synthetic derivative of quinazolinone alkaloid, previously has been shown to have anti-cancer effects in various solid and hematological malignancies. Halofuginone inhibits mainly collagen type I synthesis, and extracellular matrix formation, via the inhibition of TGFβ signaling, matrix metalloproteinase 2(MMP2), and angiogenesis. Last year, we first reported, that Halofuginone in a low doses (IC50 of 50—100 nM) induces cytotoxicity in multiple MM cell lines, including cells resistant to conventional (e.g., dexamethasone, alkylating agents, and anthracyclines) or novel (e.g. thalidomide and bortezomib) anti-MM agents and overcomes the survival and growth advantages conferred by interleukin-6, insulin-like growth factor-1 and by bone marrow stroma cells. Halofuginone induced apoptosis in a caspase 3, 8, and 9 dependent mechanisms, reduced mitochondrial membrane potential, and down regulated MCL1 protein. We now assessed the cytotoxic effect of Halofuginone in primary MM patient cells in vitro and, its effect on tumor growth and survival in in vivo models. We found that Halofuginone also induces growth inhibition and cell death in primary MM cells (n=4, IC50: 100–200nM). Importantly, Halofuginone demonstrated additive or synergistic effects with some of the established anti-MM agents such as Melphalan, Dexamethasone, and Lenalidomide. In addition, Halofuginone inhibits IL6 production in the supernatant of a co-culture of MM.1S cells with HS-5 stromal cell line. Mechanistically, Halofuginone induces MM cell death, which involves the up-regulation of c-jun NH2-terminal kinase signaling (JNK), c-Jun, as well as the p-53 proapoptotic protein. Additionally, the in vivo anti-MM activity of Halofuginone was evaluated in 2 separate in vivo models, a xenograft model in SCID mice (subcutaneous injection of MM1S cells), and a model of diffuse MM lesions in SCID-beige mice (generated by i.v. injections of OPM-2 cells). In both models, mice were first sublethally irradiated (200 rads), injected s.c or i.v., respectively, with 1×106 MM cells and then randomly assigned to receive, either treatment with 0.75mg/kg halofuginone (IP or by oral gavage, respectively; n=10) or vehicle only (n=10) on a cyclical schedule of 5 days-on/2 days-off. In both models, Halofuginone inhibited MM tumor growth and improved survival, 70% vs 40% at 140 days (NS) in the treated vs control group respectively (figure). Clinical evidence of adverse events (weight loss, vomiting) were not observed. Halofuginone may, thus represents a promising novel orally bioavailable anti-MM agent that needs further evaluation for possible clinical trials in MM. Diffuse MM lesions in SCID-beige mice. Animals were sub lethally irradiated (200rads), injected i.v. with 1×106 OPM2 cells and then randomly assigned to receive, either halofuginone treatment (by oral gavage; 0.75mg/kg (n=10) or vehicle only (n=10) on a cyclical schedule of 5 days-on/2 days-off. Figure Figure

Halofuginone, a synthetic derivative of quinazolinone alkaloid, has recently been shown to have an anti-cancer effect in various solid and hematological malignancies. It is an orally available drug with a safe toxicity profile in clinical trials of inflammatory disease and has anti-angiogenic, anti-metastatic and anti-proliferative effects in preclinical studies. It blocks TGFb signaling by targeting Smad 2/3, inhibits NFkB activity, as well as downregulates extra cellular matrix (ECM) formation via the inhibition of collagen type I formation and matrix metalloproteinase 2(MMP2). Since ECM has an important role in the bone marrow microenvironment in multiple myeloma (MM) pathogenesis, we here examined whether halofuginone induces cytotoxicity against various MM cell lines, including those sensitive and resistant to conventional and novel chemotherapies. Halofuginone (12.5–400 nM) induced cytotoxicity in a dose-dependent fashion in a variety of MM cell lines (n=20), regardless of the sensitivity to conventional chemotherapy (i.e., dexamethasone, melphalan, doxorubicin) and novel therapies (i.e., lenalidomide, bortezomib) with IC50 of 50–200 nM. In contrast, halofuginone did not induce cytotoxicity against CD40-activated peripheral blood mononuclear cells from normal donors, even at high doses (500–1000nM). Importantly, neither IL6 nor IGF1, two key growth and survival factors in MM, overcame the growth inhibitory effect of halofuginone. We further examined molecular mechanisms whereby halofuginone triggers growth inhibition in MM cells. It induced both apoptosis and necrosis in a time- (2–48h) and dose (50–200 nM) dependent manner in MM1R and OPM1 MM cells, assessed by annexin V/ PI staining. Significant dose- dependent activation of caspase 3 and 8 was demonstrated after 25–200 nM of halofuginone treatment of MM1R, MM1S, and OPM1 cells. Mitochondrial membrane potential was also reduced after 24 hours of halofuginone treatment (50, 200 nM) of MM1R and OPMI cells, as evident by the accumulation of JC1 monomers. In addition, western blot analysis showed that halofuginone induces cleavage of apoptotic proteins caspase 3/8 and PARP, and downregulates anti-apoptotic protein MCL1. Taken together, these data suggest that halofuginone has significant anti-MM activities and is a potential novel therapy in MM.