Absence Of Bone Marrow Stromal Cells Research Articles

Abstract PO-028: A high-throughput bone marrow 3D co-culture system to develop resistance to targeted agents in marginal zone lymphoma

Abstract Background. Small molecules targeting the B-cell receptor (BCR) signaling, including PI3K and BTK inhibitors, have represented a step forward for managing lymphoma patients. However, resistance often develops, negatively impacting the benefit of patients exposed to drugs. Thus, developing preclinical tools to identify modalities to overcome this phenomenon is important. The bone marrow is commonly involved in marginal zone lymphoma (MZL) patients, and it might represent a safe environment where lymphoma cells escape therapy. Bone marrow stromal cells (BMSCs) induce proliferation and drug resistance in several tumor entities, mainly through the release of cytokines. To study potential resistance mechanisms to BCR targeting agents, we aimed to develop a 3D co-culture system in which primary BMSCs were used to model the bone marrow tumor microenvironment while exposing an MZL cell line to the PI3K inhibitor copanlisib. Methods. The MZL cell line VL51 was cultured in 3D fibrin gel (10mg/mL), either alone or with primary BMSCs, in 96-well plates using the OT-2 liquid handler (Opentrons) for high-throughput seeding purposes. Copanlisib treatment started on day 3. The effects of the drug were assessed through morphological observations and quantitative measurements of images taken on days 3, 5, and 7 in the presence or absence of BMSCs. On day 7, supernatants were analyzed for 105 secreted factors using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems / Bio-Techne). Results. We observed differences in VL51 growth pattern in the different conditions. Most importantly, the co-culture with BMSCs significantly (p=0.025) decreased VL51 sensitivity to copanlisib. When not treated, VL51 cells formed significantly larger clusters (p<0.001) in the presence of BMSCs. However, upon copanlisib treatment, VL51 cells formed smaller and more distributed clusters comparable in size to those in the mono-culture. Subsequent analyses of the supernatants from the mono- or co-cultures following DMSO or copanlisib treatment identified 14 differentially secreted cytokines among the different conditions. Validation experiments were done using recombinant proteins in 2D cultures for 6 factors. Among these, recombinant IGFBP-3, serpin E1, and PTX-3 significantly decreased VL51 sensitivity to copanlisib in 2D cultures (p=0.05, 0.01, and 0.04, respectively). Validation experiments are ongoing with additional in vitro models and using BTK inhibitors. Conclusions. We created a high-throughput 3D co-culture MZL system and found that BMSCs reduce sensitivity to copanlisib in MZL models. Secretion of IGFBP-3, Serpin E1, and PTX-3 reduced response to copanlisib. This 3D-model will be used to assess resistance mechanisms to clinically relevant compounds and to evaluate alternative therapeutic approaches to improve the treatment of lymphoma patients. Citation Format: Alex Zadro, Alberto Arribas, Maria Vittoria Colombo, Eleonora Cannas, Filippo Spriano, Luciano Cascione, Chiara Arrigoni, Christian Candrian, Matteo Moretti, Francesco Bertoni. A high-throughput bone marrow 3D co-culture system to develop resistance to targeted agents in marginal zone lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-028.

Abstract B023: Enhanced survival and adherence of melphalan-induced senescent-like dormant multiple myeloma cells co-cultured with bone marrow stromal cells

Abstract Multiple myeloma (MM) is an incurable plasma cell cancer. Therapeutic advances have greatly improved patient survival; however, all patients are expected to relapse. New treatments are critically needed to overcome relapse and provide durable response. There is evidence that dormant MM cells exist in the bone marrow, where they exhibit reduced proliferative capacity and resistance to treatment. These cells are present over extended periods of time and may regain proliferative capacity to drive relapse. Tumor dormancy shares features with senescence, a state of stress-induced growth arrest. Chemotherapies, including those used in the treatment of MM, have been shown to cause therapy-induced senescence in a variety of tissues. Dormant MM cells have been shown to be maintained in their non-proliferative state by interactions with bone lining cells. Interestingly, matrix signaling has also been shown to protect against chemotherapy-induced apoptosis in an in vitro ovarian cancer organoid model. Taken together, this suggests that interaction of MM cells with the bone niche maintains growth arrest and survival. We hypothesize that therapy-induced senescence drives dormancy in MM cells that depends on interactions with the mesenchymal bone niche. GFP-expressing 5TGM1 mouse MM cells were treated with vehicle or melphalan for 48 hours and then cultured in the presence or absence of ex vivo mouse bone marrow stromal cells (BMSCs). Cells were maintained for 12 additional days to allow for the development of senescence after the initial stress response. Cells were imaged and quantified during media replacements to assess adherence/survival. Cells were then fixed and stained for nuclei, phospho-γH2AX, and telomeres by immunofluorescence/fluorescence in-situ hybridization (IF/FISH). Melphalan-treated 5TGM1 cells cultured with BMSCs were significantly growth arrested over 2 weeks compared to vehicle-treated cells. Co-culture with BMSCs significantly enhanced melphalan-treated 5TGM1 adherence compared to culture without BMSCs during media replacement. Of note, melphalan-treated 5TGM1 did not survive in the absence of BMSCs after a week in culture. Melphalan-treated 5TGM1 also exhibited significantly greater adherence with BMSCs than vehicle-treated cells. Melphalan-treated 5TGM1 were significantly larger than vehicle-treated cells and exhibited both heterochromatin foci and persistent DNA damage foci associated with telomeres, which are all markers of cellular senescence. Our findings suggest that interactions with BMSCs promote the survival of melphalan-treated MM tumor cells. The enhanced adherence of these cells also suggests that current methods for minimal residual disease assessment may be inadequate, as rare populations of MM cells adhered to the marrow stroma may not be captured by routine bone marrow aspiration. These cells exhibit features of therapy-induced senescence which may be key to MM dormancy. Thus, targeting senescence survival pathways via senolytic therapy may be a novel approach to eliminate dormant MM cells and prevent disease relapse. Citation Format: Angelo J. Guilatco, Gabriel Alvares Borges, Tamar Tchkonia, James L. Kirkland, Taxiarchis Kourelis, Matthew T. Drake, Megan Weivoda. Enhanced survival and adherence of melphalan-induced senescent-like dormant multiple myeloma cells co-cultured with bone marrow stromal cells [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B023.

Enhanced Survival and Adherence of Melphalan-Induced Senescent-like Dormant Multiple Myeloma Cells Co-Cultured with Bone Marrow Stromal Cells

Introduction: Multiple myeloma (MM) is an incurable plasma cell cancer and the second most common hematological malignancy in the United States. Therapeutic advances have greatly improved patient survival over the last decade; however, all patients are expected to relapse. New treatments are critically needed to overcome relapse and provide durable response. There is evidence that dormant MM cells exist in the bone marrow, where they exhibit reduced proliferative capacity and resistance to treatment. These cells are present over extended periods of time and may regain proliferative capacity to drive disease relapse. Tumor dormancy shares many features with senescence, a state of stress-induced growth arrest. Chemotherapies, including those used in the standard treatment of multiple myeloma such as melphalan, have been shown to cause therapy-induced senescence in a variety of tissues. Dormant MM cells have been shown to be maintained in their non-proliferative state by interactions with bone lining cells. Interestingly, matrix signaling has also been shown to protect against chemotherapy-induced apoptosis in an in vitro ovarian cancer organoid model. Taken together, this suggests that interaction of MM cells with the bone niche maintains both growth arrest and survival. We hypothesize that therapy-induced senescence drives dormancy in MM cells that is dependent on interactions with the mesenchymal bone niche. Methods: GFP-expressing 5TGM1 mouse MM cells were treated with vehicle or melphalan for 48 hours and then cultured in the presence or absence of ex vivo mouse bone marrow stromal cells (BMSCs). Cells were maintained for 12 additional days to allow for the development of a senescent phenotype after the initial stress response. During this period, cells were imaged and quantified during media replacements to assess adherence/survival. Cells were then fixed and assessed for nuclear foci, phospho-γH2AX, and telomeres by immunofluorescence/fluorescence in-situ hybridization (IF/FISH). Results: Melphalan-treated 5TGM1 cells cultured with BMSCs were significantly (p<0.0001) growth arrested over the 2-week monitoring period (mean cumulative doublings: 1.26 ± 0.179) compared to vehicle-treated cells (5.84 ± 0.122). Co-culture with BMSCs significantly (p<0.0001) enhanced melphalan-treated 5TGM1 cell adherence (mean percent of total cells washed-off: 21.7% ± 4.2%) compared to culture without BMSCs (92.2% ± 4.3%) during media replacement. Of note, melphalan-treated 5TGM1 cells did not survive in the absence of BMSCs after a week in culture. Melphalan-treated 5TGM1 cells also exhibited significantly greater (p<0.001) adherence with BMSCs (mean percent of total cells washed-off: 21.7% ± 4.2%) than vehicle-treated cells (48.9% ± 5.1%). Melphalan-treated 5TGM1 cells were significantly (p<0.0001) larger (median area in square microns: 63.96 [7.66-954.05]) than vehicle-treated cells (44.43 [7.66-434.32]) and exhibited both heterochromatin foci and persistent DNA damage foci associated with telomeres, all of which are markers of cellular senescence. Conclusions: Our findings suggest that interactions with BMSCs promote the survival of chemotherapy-treated MM tumor cells. The enhanced adherence of these melphalan-treated MM cells also suggests that current methods for minimal residual disease assessment may be inadequate, as rare populations of MM cells adhered to the marrow stroma may not be captured by routine bone marrow aspiration. These cells exhibit features of therapy-induced senescence which may be key to MM dormancy. Thus, targeting senescence survival pathways via senolytic therapy may be a novel approach to eliminate dormant MM cells and prevent disease relapse.

Long-Term Interactions with Bone Marrow Stromal Cells Alter the Molecular Dependencies of Myeloma Cells: Insights from Genomewide CRISPR-Based Functional Genomic Screens

Bone marrow stromal cells (BMSCs) play important roles in the pathophysiology of multiple myeloma (MM), other hematologic malignancies and metastatic solid tumors, by supporting the survival, proliferation and treatment resistance of malignant cells. Prior molecular profiling studies from our group and others documented that a broad range of proliferative, oncogenic and anti-apoptotic transcripts are upregulated in MM cells by their in vitro coculture with BMSCs in short-term assays (e.g. <1 week of coculture). In this study, we hypothesized that longer-term co-cultures (e.g. over 4+ weeks) with BMSCs are capable of further remodeling of the molecular networks on which MM cells depend for their survival and proliferation.To address this hypothesis, we performed a genome-wide CRISPR/Cas9-based gene editing screen, in which MM.1S cells engineered to stably express the nuclease SpCas9 were also transduced with a lentiviral particles for a pool of ~70,000 sgRNAs (Brunello library), targeting exons of ~20,000 genes (plus non-targeting control sgRNAs), under conditions of transduction which allow for an average of no more than 1 sgRNA to be incorporated in a given cell. This allowed us to convert the initial population of MM.1S cells into heterogeneous pools in which each gene is subject to individual loss-of-function, due to Cas9-mediated editing, by only 1 sgRNA (pool contains typically 4 sgRNAs per gene; 500+ cells per sgRNA; 4 replicates per experimental condition). The MM.1S-Cas9+ Brunello-transduced cells were then cultured in the absence (conventional monoculture) or presence of the immortalized human BMSC lines HS5 or HS27A (transduced with neither Cas9 nor Brunello sgRNAs), which are established BMSCs lines that can increase survival and proliferation of MM and many other tumor cell types in vitro. The monocultures or co-cultures of MM.1S cells were passaged serially over 4 and 6 weeks: each at those time points, MM cells were isolated from the cultures and processed for PCR and next generation sequencing studies to quantify the sgRNAs which exhibited depletion (vs. enrichment) in MM cells with vs. without co-culture with BMSCs.Consistent with our initial hypothesis, we observed that MM.1S cells cocultured for 4 or 6 weeks with each of the 2 BMSC lines exhibited, compared to monocultures of the same duration, statistically significant depletion (at least 3 sgRNAs/gene, MAGECK algorithm) for several genes with known or biologically plausible roles in cell survival or proliferation, including inhibitors of apoptosis, proteasome subunits, ribosomal genes, transcription factors or cofactors, regulators of splicing, autophagy and intracellular trafficking; with higher numbers of significant “hits” after 6 vs. 4 weeks of co-culture with either HS5 or HS27A. Some of these essential genes exhibited sgRNA depletion both in MM cells cultured in the absence of BMSCs and a quantitatively more pronounced sgRNA depletion in co-cultures with BMSCs, suggesting that interaction with BMSCs enhances the functional dependence of MM cells on these genes. Interestingly, some genes (e.g. with roles in translational and transcriptional elongation; mitochondrial organization and metabolism) exhibited preferential depletion of sgRNAs only in the presence, but not absence of BMSCs, suggesting that these genes represent candidate dependencies with preferential role in the context of tumor-stromal interactions. Notably, among the 2500-3000 genes with significant sgRNA depletion in the absence of stromal cells, >200 of genes (including genes involved in RNA and mRNA processing, cell cycle control, epigenetic regulation and cellular response to stress) did not exhibit sgRNA depletion in the presence of BMSCs, suggesting that the BMSC-MM cell interaction may attenuate or abrogate the role of these genes as candidate molecular dependencies in MM.Our study demonstrates the feasibility of applying functional genomic platforms, such CRISPR/Cas9, in the tumor cell compartment of co-culture systems. Our study also documents the ability of stromal co-cultures to remodel the molecular vulnerabilities of MM cells, and supports the notion that many genes play their role as dependencies and potential therapeutic targets preferentially or exclusively in the context of tumor-stromal interactions. DisclosuresMitsiades:TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Novartis: Research Funding; Takeda: Other: Employment of family member; Abbvie: Research Funding; Ono: Research Funding.

Heterogeneity of Molecular Mechanisms Determining Blood Cancer Cell Lines Resistance to Natural Killer Cells in the Context of Tumor-Stromal Interactions: Insights from Studies of Pooled “DNA-Barcoded” Cell Line Panels

Immunotherapy has remarkably changed the treatment paradigm in hematologic malignancies and natural killer (NK) cell therapy represents an attractive option, as it has been feasible and safe in early clinical trials, without graft-versus-host effects. Nevertheless, the molecular markers determining cancer cell sensitivity or resistance to NK cells, especially in the context of tumor cell interaction with the bone marrow (BM) stromal microenvironment remain incompletely understood, but have major translational relevance since these tumor-stromal interactions have been known to attenuate the response of blood cancer cells to diverse classes of pharmacological agents. To address these questions, we performed NK cell treatment of a series of cell lines from hematologic malignancies by applying a pooled "DNA-barcoded" format of these cell lines (PRISM system). We specifically quantified the dose-dependent responses to primary NK cells for 70 molecularly-annotated blood cancer cell lines, including myeloid and lymphoblastic leukemia, diffuse large B cell lymphoma and 15 multiple myeloma (MM) lines, either in presence or in absence of BM stromal cells (BMSCs) and interferon gamma (IFNg), followed by integrated computational analyses to identify candidate molecular markers correlating with tumor cell sensitivity or resistance to NK cells. NK cell cytotoxicity, quantified by the relative abundance of barcodes in treated cells compared to controls, was correlated with the transcriptional, mutational and other molecular features of each of the 70 cell lines from publicly available databases. Furthermore, data from MM cell lines were compared to our genome-wide loss of function (LOF) and gain of function (GOF) CRISPR screen data in the MM cell line MM.1S. Two distinct clusters of cell lines, sensitive and resistant to NK cell treatment, were identified. Such clusters retained distinct pattern of in vitro resistance in the presence of stroma, while showing an overall markedly decreased NK cell responsiveness, which underscores the protective effect of stromal microenvironment in blood malignancies, regardless of the addition of IFNg. RNA-seq data showed no differences in dependencies between the two clusters and no distinct gene expression patterns at baseline that clearly allows to predict NK cell response, which underscores the heterogeneity of resistance patterns at single gene level, across different hematologic malignancies. However, when comparing baseline RNAseq data to data obtained from previous GOF and LOF CRISPR screens in MM.1S, surface antigens such as PVR, ULBP1, ULBP3 were more frequently downregulated, whereas MUC1 was upregulated in resistant cells clusters. An important observation is that gene lesions such as TP53, PTEN, MMSET, commonly associated with high-risk diseases, do not affect NK cell responses in the cell lines tested. Interestingly, a gene set enrichment analysis (GSEA) showed that the cluster of resistant cells displays upregulation of class I MHC complex, class II MHC complex binding, IL7 pathway and a downregulation of transmembrane receptor protein serine/threonine kinase signaling pathway. GSEA also showed that baseline state of IFN-JAK-STAT signaling correlates with BMSCs-induced NK cell resistance, a result further confirmed by addition of IFNgto tumor-NK cocultures in the absence of BMSCs. No significant differences in NK cell response were observed when comparing cell lines of different hematologic neoplasms, suggesting that candidate markers from these studies may be relevant across different hematologic malignancies. In conclusion, this is the first study of this size correlating the molecular annotation of different concurrently-treated hematologic cell lines with their response to a NK-based treatment in the context of BMSC interaction. This study of a large panel of pooled "DNA-barcoded" cell lines provided complementary and orthogonal information to our LOF and GOF screens, expanding our potential to identify and validate molecular markers for individualized use of NK cell-based therapies in hematologic malignancies. Disclosures Mustjoki: BMS: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Research Funding. Mitsiades:EMD Serono: Research Funding; Abbvie: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding; Arch Oncology: Research Funding; Fate Therapeutics: Honoraria; Ionis Pharmaceuticals: Honoraria; Takeda: Other: employment of a relative ; Janssen/Johnson &amp; Johnson: Research Funding; TEVA: Research Funding.

Bone Marrow Stromal Cells Induce Bortezomib Resistance in Multiple Myeloma Cells through Downregulation of miRNA-101-3p Targeting Survivin

INTRODUCTIONIt is not yet fully understood how bone marrow microenvironment components especially bone marrow stromal cells (BMSCs) induce drug resistance in multiple myeloma (MM). This form of drug resistance has been suggested to pave the way for intrinsic (de novo) resistance to therapy in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Hence, deciphering the molecular mechanisms involved in induction of above resistance will help identify potential therapeutic targets in MM combined treatments. Our previous work showed that BMSCs (normal and MM patient-derived) induced resistance to bortezomib (BTZ) compared with MM cells in the absence of stroma. This resistance was associated with modulation of a transcriptome in MM cells, including prominent upregulation of oncogenes c-FOS, BIRC5 (survivin) and CCND1. However; whether these oncogenes mediate BTZ resistance in the context of BMSCs through interaction with miRNAs is not known.METHODSHuman myeloma cell lines, 8226, U266 and MM.1s, were co-cultured with MM patient-derived BMSCs or an immortalized normal human line (HS-5) in the presence of 5nM BTZ for 24 h. MM cell monocultures treated with 5nM BTZ were used as controls. Co-cultures were then applied to magnetic cell separation (EasySep, Stem Cell Technologies) to isolate MM cells for downstream analyses (western blotting and qPCR). Total RNA including miRNAs was isolated from MM cell pellets (QIAGEN miRNeasy kit), cDNAs were synthesized (QIAGEN miScript RT II kit) and applied to miScript miRNA PCR Array (SABioscience, MIHS-114ZA). After normalization of all extracted Ct values to 5 different housekeeping genes, fold changes in miRNA expression were analyzed in co-cultures compared to MM cell monocultures using the 2-ΔΔCt algorithm. Moreover, survivin gene was silenced in MM cells using Ambion® Silencer® Select siRNA and Lipofectamine RNAiMAX transfection reagent. Survivin-silenced cells were then seeded on BMSCs and exposed to BTZ. Percent apoptosis of gated CD138+ MM cells was determined using FACS. For our overexpression and 3'UTR reporter experiments, we transiently transfected MM cells with pre-miR-101-3p, scrambled miRNA or pEZX-3'UTR constructs using Endofectin reagent (all from GeneCopoeia).RESULTSBMSCs upregulated survivin gene / protein (a member of inhibitors of apoptosis family) and modulated an array of miRNAs in MM cells compared to MM cells in the absence of stroma. The more noticeably downregulated miRNAs were hsa-miR-101-3p, hsa-miR-29b-3p, hsa-miR-32-5p, hsa-miR-16-5p (4-30 fold) and highly upregulated ones included hsa-miR-221-3p, hsa-miR-409-3p, hsa-miR-193a-5p, hsa-miR-125a-5p (80-330 fold). We focused on miRNA-101-3p as it showed the highest level of downregulation (30 fold) and has been shown to function as an important tumor suppressor in other malignancies. Real time RT-PCR confirmed downregulation of miRNA-101-3p. Moreover, microRNA Data Integration Portal (mirDIP) identified miRNA-101-3p as a putative target for survivin and Luciferase activity assays confirmed binding of miRNA-101-3p to 3'UTR of survivin. In addition, overexpression of miRNA-101-3p downregulated survivin and sensitized MM cells to BTZ-induced apoptosis. Furthermore, silencing of survivin upregulated miRNA-101-3p and increased BTZ-induced apoptosis in MM cell lines both in the absence of BMSCs (Apoptosis range in BTZ-treated conditions: 57.65% ± 4.91 and 28.66% ± 0.78 for si-survivin and scrambled control, respectively, p<0.05) and in the presence of BMSCs (41.23% ± 1.43 and 14.8% ± 0.66, for si-survivin and scrambled control, respectively, p<0.05).CONCLUSIONOur results indicate that BMSCs downregulated miRNA-101-3p and upregulated survivin in MM cells compared to MM cells in the absence of stroma. Silencing of survivin or overexpression of miRNA-101-3p sensitized MM cells to BTZ in the presence of BMSCs. These findings suggest that miRNA-101-3p mediates BTZ response of MM cells in the presence of BMSCs by targeting survivin and disclose a role of survivin-miRNA-101-3p axis in regulation of BMSCs-induced BTZ resistance in MM cells, thus provide a rationale to further investigate the anti-myeloma activity of miRNA-101-3p in combination with BTZ as a potential novel therapeutic strategy in MM. DisclosuresNo relevant conflicts of interest to declare.

Targeting PYK2 Mediates Microenvironment-Specific Myeloma Cell Death

Multiple myeloma (MM) remains an all but incurable malignancy due to minimal residual disease (MRD). MRD is influenced by the survival promoting aspects of the tumor microenvironment (TME) and putative MM cancer stem cells. Therefore, identification of survival determinants specific to the TME and putative MM stem cells is critical for the design of therapy and elimination of MRD. Within the context of the TME, we have shown that collaborative signaling between β1 integrin-mediated adhesion to fibronectin (FN) and Interleukin-6 (IL-6) confers a more malignant phenotype via amplification of STAT3 activation. Further characterization of the events modulated under these culture conditions with quantitative phospho-tyrosine profiling identified 193 differentially phosphorylated peptides after adhesion to FN, treatment with IL-6, and the combination. Phosphopeptides represented proteins involved in signal transduction, cytoskeleton assembly/adhesion, transcription, RNA processing, metabolism, and the cell cycle. Seventy-seven phosphorylations were up-regulated upon adhesion, including PYK2/FAK2, paxillin, CASL, and p130CAS consistent with focal adhesion (FA) formation. Western blot analysis demonstrated PYK2 autophosphorylation of Y402 following myeloma cell adhesion to FN or bone marrow stromal cells (BMSC), correlating with the amplification of IL-6-induced STAT3 and JAK1. We hypothesized that the collaborative signaling between β1 integrin and the IL-6 signal transducer, gp130, was mediated by FA formation and PYK2. Assessing downstream gene expression, we also demonstrated that adhesion to FN alone or co-stimulation with FN and IL-6 upregulated mRNA and protein expression of the MM survival-associated gene, deptor, and this enhanced expression was abrogated by RNAi knockdown of STAT3 or PYK2. Both pharmacological and molecular targeting of PYK2 attenuated the amplification of STAT3 phosphorylation in adhered cells. Importantly, MM cells co-cultured with patient BMSCs showed similar gp130 and β1 integrin-specific enhancement of PYK2, JAK1, STAT3 signaling when adhered to patient BMSCs, demonstrating that PYK2 modulates co-stimulation specific STAT3 signaling in more complex, clinically relevant models of the microenvironment. Interestingly, targeting PYK2 with antisense oligonucleotides (ISIS Pharmaceuticals) or with the dual PYK2/FAK (Focal adhesion kinase) tyrosine kinase inhibitors VS-4718, VS-6062, and VS-6063 (Verastem, Inc.) induced preferential MM cell killing in the context of adhesion to patient BMSCs, but not in monoculture or under transwell co-culture conditions. We confirmed that this cell death correlated with inhibition of BMSC-induced PYK2 and STAT3 phosphorylation. Further analysis similarly demonstrated a preferential reduction in colony formation in BMSC-adherent MM cell lines and patient specimens treated with VS-6063 relative to MM cells in the absence of BMSCs. Co-culture with BMSCs also increased the percentage of ALDH+ MM cancer stem cells relative to monoculture. Therefore, we examined the effects of targeting PYK2 with VS-6063 in ALDH+ MM cancer stem cells. VS-6063 decreased the percentage of the ALDH+ cells associated with BMSC to greater degree than MM cells without BMSC. These data identify a novel PYK2-mediated survival pathway activated within the context of multiple microenvironmental cues, suggesting that PYK2 is a TME-specific therapeutic target. Further, putative MM cancer stem cells are sensitive to PYK2 inhibition by the FAK/PYK2 inhibitor VS-6063. Lastly, we examined the role of PYK2 in an in vivo model of the TME. Oral administration of the VS-4718 PYK2/FAK TKI twice daily significantly reduced murine myeloma disease burden as quantified by IgG2b levels and luciferase activity (p= 0.03 and 0.02). Treatment with VS-4718 also afforded a statistically significant overall survival advantage to treated mice relative to vehicle control (p=0.018). As such, these data indicate that PYK2 may be a critical target for the eradication of MRD by targeting both MM cells in the context of TME and MM cancer stem cells. These results suggest that MM represents a compelling clinical development path for FAK/PYK2 inhibitors, such as VS-4718 and VS-6063, in clinical trials focusing on eradication of MRD. DisclosuresWeaver:Verastem: Employment, Equity Ownership. Pachter:Verastem: Employment, Equity Ownership.

Targeting IL-17A in multiple myeloma: a potential novel therapeutic approach in myeloma.

We have previously demonstrated that interleukin-17A (IL-17) producing Th17 cells are significantly elevated in blood and bone marrow (BM) in multiple myeloma (MM) and IL-17A promotes MM cell growth via the expression of IL-17 receptor. In this study, we evaluated anti-human IL-17A human monoclonal antibody (mAb), AIN457 in MM. We observe significant inhibition of MM cell growth by AIN457 both in the presence and absence of BM stromal cells (BMSC). While IL-17A induces IL-6 production, AIN457 significantly down-regulated IL-6 production and MM cell-adhesion in MM-BMSC co-culture. AIN-457 also significantly inhibited osteoclast cell–differentiation. More importantly, in the SCIDhu model of human myeloma administration of AIN-457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth and reduced bone damage compared to isotype control mice. To understand the mechanism of action of anti-IL-17A mAb, we report here, that MM cells express IL-17A. We also observed that IL-17A knock-down inhibited MM cell growth and their ability to induce IL-6 production in co-cultures with BMSC. These pre-clinical observations suggest efficacy of AIN 457 in myeloma and provide the rationale for its clinical evaluation for anti-myeloma effects and for improvement of bone disease.

Targeting PYK2 Mediates Microenvironment-Specific Myeloma Cell Death

Multiple myeloma (MM) remains an incurable malignancy, at least in part, due to minimal residual disease (MRD). It has been hypothesized that MRD is influenced by the survival promoting aspects of the bone marrow tumor microenvironment (TME) and populations of putative MM cancer stem cells. Therefore, identification of survival determinants specific to the TME and putative MM stem cells is critical for the rational design of therapy and elimination of MRD1. Within the context of the TME, we have shown that collaborative signaling between β1 integrin-mediated adhesion to fibronectin (FN) and Interleukin-6 (IL-6) confers a more malignant phenotype via amplification of STAT3 activation2. We have validated the FN-adhesion-dependent amplification of IL-6-induced STAT3 phosphorylation in MM patient specimens by flow cytometry. Further characterization of the events modulated under these culture conditions with quantitative phospho-tyrosine profiling identified 193 differentially phosphorylated peptides after adhesion to FN, treatment with IL-6, and the combination. Phosphopeptides represented proteins involved in signal transduction, cytoskeleton assembly/adhesion, transcription, RNA processing, metabolism, and the cell cycle. Seventy-seven phosphorylations were up-regulated upon adhesion, including PYK2/FAK2, paxillin, CASL, and p130CAS consistent with focal adhesion (FA) formation. Western blot analysis demonstrated PYK2 autophosphorylation of Y402 following myeloma cell adhesion to FN or bone marrow stromal cells (BMSC), correlating with the amplification of IL-6-induced STAT3 and JAK1. We hypothesized that the collaborative signaling between β1 integrin and the IL-6 signal transducer, gp130, was mediated by FA formation and PYK2. Although PYK2 has been linked to downstream signaling events of other JAK-dependent cytokine receptors, it has not been associated with a STAT3 amplification mechanism within the Type I or Type II cytokine receptor families3-6. Assessing downstream gene expression, we also demonstrated that adhesion to FN alone or co-stimulation with FN and IL-6 upregulated mRNA and protein expression of the MM survival-associated gene, deptor7, and this enhanced expression was abrogated by RNAi knockdown of STAT3 or PYK2. Both pharmacological and molecular targeting of PYK2 attenuated the amplification of STAT3 phosphorylation in adhered cells. Importantly, MM cells co-cultured with patient BMSCs showed similar gp130 and β1 integrin-specific enhancement of PYK2, JAK1, STAT3 signaling when adhered to patient BMSCs, demonstrating that PYK2 modulates co-stimulation specific STAT3 signaling in more complex, clinically relevant models of the microenvironment. Interestingly, targeting PYK2 with antisense oligonucleotides (ISIS Pharmaceuticals) or with the dual PYK2/FAK (Focal adhesion kinase) tyrosine kinase inhibitors VS-4718, VS-6062, and VS-6063 (Verastem, Inc.) induced preferential MM cell killing in the context of adhesion to patient BMSCs, but not in monoculture or under transwell co-culture conditions. We confirmed that this cell death correlated with inhibition of BMSC-induced PYK2 and STAT3 phosphorylation. Further analysis similarly demonstrated a preferential reduction in colony formation in BMSC-adherent MM cell lines and patient specimens treated with VS-6063 relative to MM cells in the absence of BMSCs. Lastly, we examined the effects of targeting PYK2 with VS-6063 in ALDH+ MM cancer stem cells. We demonstrated that co-culture with BMSCs increased the percentage of ALDH+ MM cancer stems relative to monoculture. VS-6063 decreased the percentage of the ALDH+ cells associated with BMSC to greater degree than MM cells without BMSC. These data identify a novel PYK2-mediated survival pathway activated within the context of multiple microenvironmental cues, suggesting that PYK2 is a putative TME-specific therapeutic target. Further, putative MM cancer stem cells are sensitive to PYK2 inhibition by the FAK/PYK2 inhibitor VS-6063. As such, these data indicate that PYK2 may be critical target for the eradication of MRD by targeting both MM cells in the context of TME and MM cancer stem cells. These results suggest that MM represents a compelling clinical development path for FAK/PYK2 inhibitors, such as VS-4718 and VS-6063, in clinical trials focusing on eradication of MRD. DisclosuresShain:Onyx/Amgen: Research Funding; Celgene: Research Funding; Envision/Celgene: Speakers Bureau; L&M Healthcare/Onyx/Amgen: Speakers Bureau. Ring:Verastem: Employment. Pachter:Verastem Inc.: Employment, Equity Ownership.

Tumor Cell-Dependent Differences in Stroma-Induced Changes to Bortezomib Sensitivity.

Abstract 2468In 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<0.02, log-rank tests) after treatment with bortezomib. These observations highlight the notion that tumor cell responses to a given agent in the presence of non-malignant stromal cells can exhibit substantial qualitative and quantitative variation, depending on the specific tumor cell type tested, as well as the particular stromal cell population and conditions of the co-culture. Our findings highlight the need to apply combinatorial high-throughput scalable platforms, such as CS-BLI, to evaluate the different permutations of interactions between tumor cells, non-malignant accessory cells of the microenvironment and administered therapeutics. This study also provides a comprehensive functional oncogenomic framework to identify prognostically relevant molecular mediators of stroma-induced resistance to therapy in MM. 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.

In Vitro Anti-Myeloma Activity of the Multitargeted Kinase Inhibitor Midostaurin in the Context of Heterotypic Cocultures of Myeloma Cells with Nonmalignant Microenvironmental Accessory Cells

Abstract 2923Midostaurin (PKC412; Novartis Pharmaceuticals) is a multi-targeted kinase inhibitor currently being evaluated in clinical trials in acute myelogenous leukemia (AML), because of its potent activity in cells expressing mutant FLT3. Prior preclinical studies from our groups have shown that PKC412 has FLT3-independent anti-MM activity, and the effects on AML cells is suppressed by the presence of conditioned media from bone marrow stromal cells (BMSCs), such as the immortalized BMSC line HS-5 (Weisberg et al. Mol Cancer Ther 2007). In this study, we evaluated whether the microenvironment-dependent drug resistance to PKC412 applies to not only AML cells, but also to cells from MM and other FLT3-negative malignant cells. We tested a panel of cells from MM (n=8), FLT-ITDneg AML (n=1), CML (n=2) and breast cancer (n=2) for their response to PKC412 in the presence or absence of BMSCs and other non-malignant accessory cells using tumor cell compartment-specific bioluminescence imaging (CS-BLI), as in our antecedent studies (McMillin et al. Nat Medicine 2010). We also compared the PKC412 response of the aforementioned neoplastic cells when cultured in vitro in the presence or absence of conditioned media (CM) from different types of BMSCs known to confer PKC412 resistance in FLT3-mutant AML cells. Consistent with our previous studies of PKC412 treatment in conventional cultures of MM cells in isolation, we observed that PKC412 exhibits an anti-proliferative effect within the first 24 hrs of treatment, with major reduction of the numbers of viable cells at 48 and 72 hrs. At sub-micromolar doses that did not significantly affect the viability of non-malignant accessory cells tested, PKC412 had similar (or for some MM cell lines had more pronounced) activity against the MM cells, both in the presence and absence of the non-malignant accessory cells tested (HS-5, HS-27a, NIH-3T3 cells with or without transfection with human CD40L, etc.). In contrast, under the same experimental conditions, coculture with either BMSCs or exposure to their conditional media, decreased the response of MM cells to dexamethasone. These results suggested in contrast to the impact on FLT3mut AML, that the anti-MM activity of PKC412 is preserved (and in some cases slightly enhanced) when the MM cells interact with microenvironmental accessory cells and/or their secreted growth/survival factors. To obtain insight on possible mechanistic foundations of these observations, we examined the pattern of kinases inhibited by PKC412 at sub-μM concentrations (using FLT3 and FGFR3, known targets of PKC412 as positive controls). The results of in vitro kinase activity assays showed that PKC412 potently suppresses the aforementioned positive controls, but also exerts >50% inhibitory effect on the in vitro activity of additional kinases such as Akt2, Pim1, GSK3a, PDK1, p70S6K, SRC and Aurora A. Many of these kinases are known to participate in proliferative/anti-apoptotic signaling cascades downstream of cytokine/growth factor receptors or cell adhesion-mediated events triggered during MM–stromal interactions. We therefore conclude that the influence of the tumor microenvironment on the anti-neoplastic effects of PKC412 may be tumor-type dependent. The anti-MM activity of PKC412 is not subject to drug resistance triggered by non-malignant accessory cells, and conversely is occasionally moderately enhanced by these MM-stromal interactions. Mechanistically, this observation may be attributed in part to the multi-targeted nature of this inhibitor and, in particular, its aggregate impact on several kinases known to mediate stroma-induced proliferative and antipoptotic signaling in MM. Disclosures:Griffin:Novartis Pharmaceuticals: Consultancy, Research Funding. Richardson:Millennium: ; Celgene: ; Johnson & Johnson: ; Novartis: ; Bristol Myers Squibb:. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Mitsiades:EMD Serono: Research Funding; AVEO Pharma: Research Funding; Amgen: Research Funding; OSI Pharmaceuticals: Research Funding; PharmaMar: licensing royalties; Amnis Therapeutics: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding.

Blocking the ZZ Domain of Sequestosome 1/p62 Suppress the Enhancement of Myeloma Cell Growth and Osteoclast Formation by Marrow Stromal Cells

Abstract 888The marrow microenvironment enhances both tumor growth and bone destruction in multiple myeloma (MM) through MM cell-induced activation of multiple signaling pathways in bone marrow stromal cells (BMSC) by TNFα. We reported that sequestosome-1 (p62) acts as a signaling hub for NF-kB, MAPK and PI3K activation in BMSC of MM patients and enhances MM growth and osteoclast (OCL) formation. p62 is composed of 5 domains that are involved in protein–protein interactions required for formation of these signaling complexes, but which domain of p62 mediates increase MM growth and OCL formation is unclear. Therefore, deletion constructs of p62 that lacked each of the 5 domains (PB1, ZZ, p38, TBS or UBA) were transfected into a p62−/− stromal cell line. We found that the ZZ domains mediated BMSC enhancement of MM cell growth, IL-6 production, VCAM-1 expression and OCL formation. Using virtual modeling of the ZZ domain, we identified 6 candidate p62-ZZ inhibitory molecules and tested them for their capacity to block enhanced MM cell growth, OCL formation, IL-6 production, and VCAM-1 expression on BMSC induced by TNFα.When MM1.S, RPMI8266, ANBL6 MM cell-lines or CD138+ primary MM cells were cultured in the absence of BMSC with p62-ZZ inhibitor #3 (10mM), this inhibitor directly induced cell death. The p62-ZZ inhibitor-induced cell death was characterized by an increase in reactive oxygen species (ROS) production by inhibiting NF-kB activation, and apoptotic cell death by triggering the activation of caspases 3, 7, and 9. This inhibitor had an IC50 of 4.6mM for MM1.S survival. In contrast, CFU-Blast formation by human CD34+ cells was not inhibited by p62-ZZ inhibitor #3 (10mM). p62-ZZ inhibitor #3 (10mM) treatment of human OCL precursors derived from CFU-GM, inhibited OCL formation by blocking precursor proliferation. To examine the specificity of the p62-ZZ inhibitor #3, we tested its effects on OCL formation by CD11b+ mononuclear cells from wild type (WT) and p62−/− mice cultured with TNFα for 7 days. p62-ZZ inhibitor #3 blocked WT OCL formation but did not block p62−/− OCL formation. The p62-ZZ inhibitor also blocked VCAM-1 expression and IL-6 production by normal and MM patient stromal cells induced by TNFα compared to vehicle. Importantly, the inhibitor (10mM) did not block stromal cell proliferation. Further, the inhibitor blocked TNFα induced PKCζ phosphorylation in stromal cells and MM1.S when the cells were pretreated with the p62-ZZ inhibitor for 3 hours. These results demonstrate that p62-ZZ inhibitor #3 specifically blocks both stromal cells independent and dependent MM cell growth and OCL formation but does not affect hematopoietic or stromal cell growth. These results support p62 as a potential novel therapeutic target for MM. Disclosures:Roodman:Amgen: Consultancy; Millennium: Consultancy.

Bone Marrow Niche Down-Regulates Mir-30 In Multiple Myeloma Cells to Promote Cancer Progression and Cancer Initiation by Targeting BCL9/Wnt Pathway.

AbstractAbstract 1569“Niche” is a specialized microenvironment which controls the fate specification and development of stem and progenitor cells. The bone marrow (BM) niche is composed of osteoblasts, osteoclasts, bone marrow endothelial cells, stromal cells, adipocytes, and extracellular matrix proteins (ECM). These elements provide an optimal growth environment for multiple hematological malignancies, including multiple myeloma (MM) cancer stem cells (CSCs). For example, the MM bone marrow stromal cells (BMSCs) confer survival and chemoresistance of MM cells to current therapies. A better and more detailed understanding of the neoplastic MM niche will therefore provide a model for identifying and validating novel targeted therapies directed against MM. Our previous data in 78 MM patient samples showed that miR-30 family members were more weakly expressed in patients samples compared with normal plasma cells. In our present study, we showed that miR-30 is downregulated by co-culture of either MM cell line or MM patients sample with BMSCs. Bioinformatics analysis showed that Bcl9 is a common target of miR-30, with two different binding sites in 3'UTR region of Bcl9 mRNA predicted by 3 different web-based softwares. Importantly, we confirmed Bcl9 as a direct target of miR-30 in MM cells by both gain of function and loss of function studies. Specifically, ectopic expression of miR-30 by using HIV based lentivirus (V-miR-30) downregulates Bcl9 gene expression by mRNA degradation in either 293T or H929 cells compared with GFP control cells (V-GFP). Conversely, knockdown of miR-30 family expression upregulates Bcl9 mRNA and protein level in MMS1 cells, which have relatively higher miR-30 and lower Bcl9 levels. To prove a direct interaction between miR-30 and the 3'UTR of Bcl9, two wild type Bcl9-3'UTR reporter vectors (pmiR-Bcl9-30-1W and pmiR-Bcl9-30-2W) and two mutant Bcl9-3'UTR reporter vectors (pmiR-Bcl9-30-1M and pmiR-Bcl9-30-2M) were cotransfected into H929 cells, together with V-miR-30 or V-GFP. Luciferase activity of wild type, but not mutant, was significantly decreased with V-miR-30 with respect to V-GFP. Downregulation of miR-30 induced overexpression of Bcl9 in MM cell line or patients samples, which in turn transcriptionally activates WNT pathway downstream target genes such as Axin2 and CD44. As expected, WNT pathway TOP/FOP luciferase activity was induced by knockdown miR-30 in MMS1 cells and suppressed by ectopic expression of miR-30 in H929 cells. In addition, the stem cell population of H929 V-miR-30 dramatically decreased, identified using functional Hoechst 33342 stem cell staining assay. Moreover, in experiments using stem cell medium to culture sorted CSCs, the sphere number and size is less in H929 V-miR-30c CSCs compared with H929 V-GFP control CSCs. In addition, proliferation and tumor formation were inhibited in vitro and in vivo in H929 V-miR-30 stable cells compared with H929 V-GFP cells. Finally, H929 V-miR-30 stable cells were more sensitive to bortezomib treatment than GFP control cells in the presence or absence of BMSCs. Our studies therefore demonstrate that miR-30 regulates the WNT pathway by targeting Bcl9 in MM cells in the BM milieu and represents a promising novel therapeutic target in MM. Disclosures:Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Human Monoclonal Antibody Targeting IL-17A (AIN457) Down-Regulates MM Cell-Growth and Survival and Inhibits Osteoclast Development In Vitro and In Vivo: A Potential Novel Therapeutic Application In Myeloma

AbstractAbstract 456We have previously demonstrated that IL-17 producing TH17cells, a new subset of T helper cells, are significantly elevated in peripheral blood and bone marrow (BM) from patients with multiple myeloma (MM) and IL-17 produced by these cells promotes MM cell growth and survival, suppresses immune responses and induces osteoclast differentiation both in vitro and in vivo. Based on these observations we have investigated the effects of human anti-IL-17A monoclonal antibody (mAb), AIN-457, in MM. We observed that whereas IL-17A induced proliferation of MM cells (+30.7+2.7%) compared to control; anti-IL-17A mAb AIN-457 significantly inhibited MM cell growth both in presence and absence of BM stromal cells, as measured by thymidine incorporation (−18.7+1.5% and −22.7+2.6% respectively). We have further confirmed these inhibitory effects of anti-IL-17A antibody using MM cell colony forming assay with MethoCult agar plates. While presence of IL-17A increased the colony number from 80 in control plates to 188, presence of AIN-457 reduced the colonies to <40 per unit area (p < 0.01). Evaluating the mechanism of action, IL-17A induced IL-6 production (+289.6+38%; p<0.01); while AIN457 significantly down-regulated IL-6 production (−25+7%; p<0.05) in MM-BMSC co-culture. We also observed that AIN-457 significantly reduced adhesion of MM cells to stromal cells (27%, p=0.011). AIN457 significantly inhibited IL-6 production in human fetal bone chips in the presence of MM cells within 24 hours of ex-vivo culture (control − 487+39 pg/ml; IL-17 990+27 pg/ml; p<0.01 and AIN-457 − 326+7 pg/ml; p<0.01). Since IL-17A plays a critical role in bone damage, we further evaluated the effect of this mAb on the generation of osteoclasts. When normal BM cells were cultured for three weeks in osteoclast supporting medium, presence of AIN-457 significantly inhibited TRAP+ multinucleated osteoclast cell numbers by>60%. We next evaluated the efficacy of AIN-457 in vivo in the murine models of human myeloma; in the subcutaneous MM xenograft model, we observed significant reduction in tumor volumes by pre-treatment with AIN457 compared to control (142+77 mm versus 355+56 mm, p=0.019) while IL-17A significantly increased MM cell growth (727+135 mm, p=0.01). More importantly in the SCIDhu model of human myeloma where MM cells grow within the human microenvironment in the mice, administration of AIN-457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth as measured by human sIL-6 receptor compared to control mice (5.9±2.2 ng/ml versus 23.2±6.3 ng/ml; n=7; P <0.01). These pre-clinical in vitro and in vivo observations confirm the role of IL-17A produced by TH17 cells in MM and provide the rationale for clinical evaluation of AIN 457 for both anti-myeloma effects as well as to improve bone disease in myeloma. Disclosures:No relevant conflicts of interest to declare.

Daratumumab, a Novel Potent Human Anti-CD38 Monoclonal Antibody, Induces Significant Killing of Human Multiple Myeloma Cells: Therapeutic Implication.

Abstract 608CD38 is a type II transmembrane glycoprotein highly expressed in many hematological malignancies including multiple myeloma (MM), representing a promising target for monoclonal antibody (mAb)-based targeted immunotherapy. We here investigated the mechanisms of anti-MM activity of daratumumab, a novel anti-CD38 mAb currently in clinical development, to treat human MM. Daratumumab was generated in human Ig transgenic HuMab-mice® and was selected for its excellent binding characteristics. It strongly reacts against a panel of CD38-expressing MM cell lines (>94%, 17/18) that are sensitive or resistant to current treatments for MM: median fluorescence intensity (MFI) observed was 378-20764 and 15-140 for daratuzumab and isotype control Ab, respectively. U266 cells, which lack CD38 mRNA, did not bind daratuzumab. Significantly, daratuzumab induced NK-mediated antibody-dependent cellular cytotoxicity (ADCC) against CD38-expressing MM cells but not CD38-negative U266 cells, confirming its specific targeting. Daratuzumab-mediated MM cell lysis was dose-dependent, with maximum lysis of the CD38-expressing MM cell lines (n=17) at concentrations of 0.01-0.1μg/ml. We also determined potential effects of daratumumab on NK and peripheral blood mononuclear cells (PBMC) effector cells, since these cells express CD38, albeit at lower levels than MM cells. Overnight pretreatment of PBMCs or purified NK cells from 2 normal donors with daratumumab (2 μg/ml) did not significantly alter daratumumab-induced ADCC against MM1S target cells. The bone marrow (BM) microenvironment has been shown to protect MM cells against apoptosis and stimulate their growth; we therefore next asked whether daratumumab effectively induced ADCC against MM cells in the presence of BM stromal cells (BMSCs). Daratumumab induced a similar, dose-dependent, ADCC of dexamethasone-sensitive MM1S as well as dexamethasone-resistant MM1R cells both in the presence and absence of BMSCs, suggesting that daratumumab could indeed effectively induce ADCC in the BM microenvironment. Significantly, our findings translated to patient cells, since daratumumab induced ADCC of both allogeneic (n=2) and autologous (n=9) patient MM cells. We next turned our attention to complement-mediated cytotoxicity (CDC). In calcein AM-labeling assays in the presence of normal human serum, daratumumab induced dose-dependent CDC of LP-1 and XG-1 MM cells expressing low levels of CD59 and CD55 both in the absence or presence of BMSCs. Effective daratumumab-induced CDC of CD138-purified MM cells was seen for 6 of 9 patients. Daratumumab-induced CDC was not affected by the expression of complement inhibitor proteins in this cohort of patients. Finally, daratumumab induced apoptosis in Ramos and Daudi cells when crosslinked by anti-IgG antibody, and it induced cell death in CD138-purified patient MM cells, as assessed by annexin V/PI staining. Taken together, our results indicate that daratumumab is able to effectively kill patient MM tumor cells via several cytotoxic mechanisms, supporting clinical development of daratumumab to improve patient outcome in MM. Disclosures:de Weers:Genmab: Employment. Bakker:Genmab: Employment. Vink:Genmab: Employment. Jacobs:Genmab: Employment. Oomen:Genmab: Employment. Bleeker:Genmab: Employment. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. van de Winkel:Genmab: Employment. Parren:Genmab: Employment. Richardson:Keryx Biopharmaceuticals: Honoraria. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

The Role of FGFR in the Progression of Waldenstrom's Macroglubulinemia and the Effect of Its Inhibition by TKI-258.

Abstract 3737Poster Board III-673 INTRODUCTIONWaldenström macroglobulinemia (WM) is a low grade non-Hodgkin lymphoma, characterized by the presence of abnormal lymphoplasmacytic cells producing high levels of IgM. Although indolent, WM remains incurable, and therefore, there is an urgent need for rationally designed therapy in WM. Receptor tyrosine kinases (RTKs) are cell surface receptors for growth factors, cytokines and hormones which have a critical role in the development and progression of many types of cancer. However, their role in WM was not identified. TKI-258 (Novartis, Basel, Switzerland) is an ATP-competitive inhibitor with activity against (multiple) receptor tyrosine kinases including FGFR and other RTKs. We hypothesized that FGFR is up-regulated in WM and plays a major role in its progression; and that TKI-258 would reduce tumor progression in WM. METHODS AND RESULTSWe tested the expression of FGFR3 on WM cells and found overexpression of this RTK compared to CD19+ cells from healthy donors. The activation of FGFR3 by recombinant FGF induced MAPK signaling pathway in WM cells including phosphorylation of RAF, ERK and STAT3. Also it induced PI3K signaling including phosphorylation of AKT, S6R and GSK3. TKI-258 inhibited the FGF induced activation of the MAPK and PI3K signaling pathways in a dose- response manner. Using MTT assay we tested the effect of TKI-258 ( 0 to 2.5 uM) on the survival of WM cell line BCWM-1, on IgM secreting cell line MEC-1, and on CD19+ cells selected from WM patient sample. We found that the TKI-258 induced cell death in all sample tested with an IC50 ranging 0.8-1 uM. Testing the effect of TKI-258 on the survival of CD19+ cells selected from peripheral blood or mononuclear cell from healthy donors showed a minimal effect of less than 10% cell death. These results provide a wide therapeutic window for the use of TKI-258 in WM. Moreover, we tested the effect of TKI-258 on the apoptosis of WM cells by flow cytometry using the apoptosis marker APO-2.7, and found that TKI-258 induced apoptosis of WM cells in a dose-response manner at both 24 and 48 treatment. Moreover these results were confirmed by testing changes in the expression of apoptosis related proteins in response to TKI-258 by immunoblotting, including induction of PARP, and caspase-3 and caspase-9 cleavage. In correlation with these results, cell cycle analysis by PI staining and analysis by flow cytometry of WM cells treated with TKI-258 for 24 hrs showed induction of sub-G1 increase in a dose response manner with an IC50 about 1uM. To test the effect of TKI-258 on the interaction of WM cells with the microenvironment we examined the effect of TKI-258 on adhesion of WM cells to fibronectin and bone marrow stromal cells (BMSCs), and found that TKI-258 induced a 50% decrease of adhesion. Moreover, we found no effect on the chemotaxis of WM induce by stroma derived factor-1 (SDF1). To test the direct effect of TKI-258 on the interaction with the microenvironment, we examined the proliferation of WM when cultured alone of in co-culture with BMSCs by 3H-thymidine uptake assay. We showed that TKI-258 inhibited the proliferation of WM cells with an IC50 of 0.8 uM, in the presence or absence of BMSCs. CONCLUSIONIn conclusion, we found an overexpression of FGFR3 in WM cells compared to CD19+ cells from healthy donors, and that TKI-258 inhibited the activation of proliferative pathways induced by activation of FGFR3 and led to inhibition of proliferation and apoptosis of WM cells. Disclosures:Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Promoting Osteoblastogenesis Using a Novel Dkk-1 Neutralizing Antibody in the Treatment of Multiple Myeloma Related Bone Disease

Treatment of bone disease in multiple myeloma (MM) has largely focussed on the osteoclast axis while the osteoblast axis has been underexploited. Dkk1, an inhibitor of the wingless int (wnt) pathway, is important in osteoblastogenesis. Increased expression of Dkk1 in a subset of MM patients and its association with lytic bone disease opens up the potential of targeting the osteoblast axis. The aim of this study was to test the effect of a Dkk-1 neutralizing chimeric antibody (Mab B3) on osteoblasts (OB), osteoclasts (OC) and MM cells in the context of the bone microenvironment. First, we tested the expression of Dkk1 in plasma and bone marrow of 16 MM patients and 10 MM cell lines. Dkk1 levels were >18 ng/mL in 2 out of 16 patients; levels were comparable in blood and bone marrow plasma. In contrast, very little Dkk1 (2–9 ng/ml) was produced by bone marrow stromal cells (BMSC). One out of 10 MM cell lines (INA-6) expressed low concentrations of Dkk1 in the supernatant. Next, we tested the effect of Mab B3 on MM cell lines, in the presence or absence of BMSC, and on OB and OC from MM patient derived bone marrow. The effects on OC were evaluated by TRAP staining and pit formation. Effects on OB were assayed by alkaline phosphatase staining and alizarin red assays for calcium deposition. Mab B3 treatment did not demonstrate direct cytotoxic effects on MM cell lines negative for Dkk1. Mab B3, however, enhanced OB differentiation and calcium deposition in a dose dependent manner and inhibited OC differentiation and function, as evidenced by a decrease in number of multinucleated TRAP+ cells and a decrease in pit formation. Ongoing studies are addressing the effect of Mab B3 on MM cells in the context of OC and OB. Mab B3 is also undergoing in vivo testing in a SCID-hu model bearing INA-6 MM cells. These studies and the underlying mechanism of action of Mab B3 will be presented. Our preliminary data suggests that Mab B3 has anabolic bone effects; a corresponding human monoclonal antibody may be useful for the treatment of MM related bone disease. Future studies will evaluate Mab B3 in combination with catabolic agents such as bisphosphonates with the goal of restoring normal bone homeostatsis.

Development of a Model for Evaluating the Interaction Between Human Pre-B Acute Lymphoblastic Leukemic Cells and the Bone Marrow Stromal Cell Microenvironment

Clonal expansion of B-cell precursor acute lymphoblastic leukemia (ALL) is potentially regulated by survival, growth, and death signals transduced by the bone marrow (BM) microenvironment. Using a human BM stromal cell culture that supports the growth of normal human B-cell precursors, we established a pre-B ALL cell line designated BLIN-2. BLIN-2 has a clonal rearrangement of the Ig heavy chain locus, a dic(9;20) chromosomal abnormality, and a bi-allelic deletion of thep16INK4a and p19ARF genes. The most interesting feature of BLIN-2 is an absolute dependence on adherent human BM stromal cells for sustained survival and growth. BLIN-2 cultured in the absence of BM stromal cells undergo apoptosis, and direct contact with viable BM stromal cells is essential for optimal growth. BLIN-2 cells also grow on vascular cell adhesion molecule-1 (VCAM-1)–negative human skin fibroblasts, making it unlikely that a very late antigen-4 (VLA-4)/VCAM-1 interaction is required for BLIN-2 growth. Western blot analysis of BLIN-2 cells cultured in the presence or absence of BM stromal cells demonstrates that contact of BLIN-2 with BM stromal cells induces hyperphosphorylation of Rb. In contrast, the pre-B ALL cell line BLIN-1, which has a bi-allelic deletion of p16INK4ap19ARF but does not require BM stromal cells for growth, does not undergo Rb phosphorylation after BM stromal cell contact. The BLIN-2 cell line will facilitate identification of ligand/receptor interactions at the B-cell precursor/BM stromal cell interface and may provide new insight into microenvironmental regulation of leukemic cell survival and growth.

Development of a Model for Evaluating the Interaction Between Human Pre-B Acute Lymphoblastic Leukemic Cells and the Bone Marrow Stromal Cell Microenvironment

Clonal expansion of B-cell precursor acute lymphoblastic leukemia (ALL) is potentially regulated by survival, growth, and death signals transduced by the bone marrow (BM) microenvironment. Using a human BM stromal cell culture that supports the growth of normal human B-cell precursors, we established a pre-B ALL cell line designated BLIN-2. BLIN-2 has a clonal rearrangement of the Ig heavy chain locus, a dic(9;20) chromosomal abnormality, and a bi-allelic deletion of the p16(INK4a) and p19(ARF) genes. The most interesting feature of BLIN-2 is an absolute dependence on adherent human BM stromal cells for sustained survival and growth. BLIN-2 cultured in the absence of BM stromal cells undergo apoptosis, and direct contact with viable BM stromal cells is essential for optimal growth. BLIN-2 cells also grow on vascular cell adhesion molecule-1 (VCAM-1)-negative human skin fibroblasts, making it unlikely that a very late antigen-4 (VLA-4)/VCAM-1 interaction is required for BLIN-2 growth. Western blot analysis of BLIN-2 cells cultured in the presence or absence of BM stromal cells demonstrates that contact of BLIN-2 with BM stromal cells induces hyperphosphorylation of Rb. In contrast, the pre-B ALL cell line BLIN-1, which has a bi-allelic deletion of p16(INK4a) p19(ARF) but does not require BM stromal cells for growth, does not undergo Rb phosphorylation after BM stromal cell contact. The BLIN-2 cell line will facilitate identification of ligand/receptor interactions at the B-cell precursor/BM stromal cell interface and may provide new insight into microenvironmental regulation of leukemic cell survival and growth.

Human Pre-B Cells Differentiate Into Ig-Secreting Plasma Cells in the Presence of Interleukin-4 and Activated CD4+ T Cells or Their Membranes

AbstractStudies on human B-cell development have been hampered by the lack of reproducible culture techniques to induce pre-B cells to differentiate into Ig-secreting plasma cells. Here, we describe that highly purified surface (s) µ-, cytoplasmic (c)µ+. CD10 +, CD19 + human pre-B cells derived from fetal bone marrow (BM) differentiate with high frequencies into Ig-secreting plasma cells, when cocul-tured with activated, cloned CD4+ T cells and with interleukin-4 (IL-4). Production of IgM, total IgG, lgG4, and IgE in pre-B-cell cultures was detected, indicating that the cells also underwent Ig isotype switching. Pre-B-cell differentiation occurred in the absence of BM stromal cells, IL-7, and stem cell factor (SCF). However, IL-7 significantly enhanced the levels of Ig produced, whereas SCF was ineffective. Neutralizing anti—IL-4 monoclonal antibodies (MoAbs) completely inhibited pre-B—cell differentiation showing the specificity of the reaction. Intact CD4+ T-cell clones could be replaced by membrane preparations of these cells, indicating that the costimulatory signals provided by the activated CD4+ T cells are contact-mediated. In contrast, anti-CD40 MoAbs failed to provide the costimulatory signal required for pre-B—cell differentiation, which may be related to the very low expression of CD40 on fetal BM B cells. Activated CD4+ T cells and IL-4 also induced sµ expression and Ig synthesis in cultures initiated with pre-B cells that had been preincubated in medium for 2 days, and from which spontaneously emerging sµ+ B cells were removed by using a fluorescence-activated cell sorter. These results support the notion that the Ig synthesis observed in pre-B-cell cultures was not caused by outgrowth and differentiation of cells that spontaneously matured into sµ+ B cells. In addition, IL-4 and CD4+ T cells strongly enhanced CD40 and HLA-DR expression on the majority of cultured pre-B cells, further indicating that CD4+ T cells and IL-4 activate bona fide pre-B cells. Taken together, these data indicate that activated CD4+ T cells and IL-4 can provide all the necessary signals required for human pre-B cells to differentiate into Ig-secreting plasma cells.