Role of Hypoxia in the Progression and Dissemination of Multiple Myeloma.

Abstract Abstract 421 INTRODUCTION: Multiple myeloma (MM) is characterized by the disseminated involvement of the bone-marrow (BM), and its progression involves a continuous circulation of the MM cells in the peripheral blood and homing back to the BM. Several reports have described the mechanism involved in homing of MM cells to the BM. However, the driving force to metastasize from one site of the BM to another is not yet understood. Hypoxia (oxygen deprivation) was associated with poor patient prognosis in solid tumors, and several studies have shown that BM has a hypoxic nature. We hypothesize that the hypoxic nature of the BM and the rapid development of MM may cause induction of hypoxia in the tumor microenvironment, induce stress in the MM cells, and drive them to disseminate to new BM niches with normal oxygen levels (normoxia). METHODS AND RESULTS: First we have tested the level of oxygenation of MM cells and cells from its BM microenvironment by injection of pimonidazole (PIM) to mice with MM and determined the levels of PIM binding in MM cells and stromal cells in the microenvironment by flow cytometry. The whole cells population in the BM was hypoxic, while the MM cells were more hypoxic compared to cells in the microenvironment. We have mimicked these results in vitro by incubation of MM cells (cell lines and patients samples) in hypoxic conditions (0.5% oxygen, for 24 hrs). Induction of hypoxia was also verified by detection of increased binding of PIM by flow cytometry, and increased levels of HIF1 and HIF2 in MM cells by immunoblotting. MTT assay showed that hypoxia significantly decreased (40%) the survival of MM cells. Immunoblotting showed a downregulation of proliferative signaling pathway, including PI3K, AKT, mTOR and ERK; and induction of stress pathways including MKK-3 and 6, and p38. Apoptosis was not detected by flow cytometry after 24 hrs of hypoxic conditions. These results were confirmed by immunoblotting, which showed no change in apoptosis related proteins including caspases 3 and 7, Bcl-2, Bcl-xL and Mcl-1. However, hypoxia induced G1-phase cell cycle arrest (increase of 20% in G1)., These results were confirmed by immunoblotting showing downregulation of proteins associated with G1 to S phase transition including cyclins D1, D2, D3 and E1, and pRb; and upregulation of cell cycle inhibitor p27. Testing the effect of hypoxia on the adhesion properties of MM cells to BM stromal cells (BMSCs) revealed that hypoxic MM cells were 50% less adherent to BMSCs compared to normoxic MM cells. Also, hypoxic BMSCs induced 30% less adhesion of MM cells compared to normoxic BMSCs. Mechanistically, immunoblotting showed a significant decrease in the expression of cadherins in both hypoxic MM cells and hypoxic BMSCs. The chemokine stromal-cell derived factor-1-alpha (SDF1) is known to increase adhesive properties of MM cells, and to induce MM retention in the BM. Testing the levels of SDF1 revealed that hypoxia decreased SDF1 secretion (40%) from BMSCs. As a result, media from hypoxic BMSCs induce significantly decreased MM cell chemotaxis (20%) compared to media from normoxic BMSCs. These findings suggest that hypoxic BM will retain less MM cells in the BM and allow more egression. Comparison of the ability of hypoxic and normoxic MM cells to migrate towards normoxic BMSCs or SDF1 revealed that hypoxic MM cells had increased (250% and 350%, respectively) chemotactic properties compared to normoxic MM cells. These findings correlated with the increased expression of CXCR4 on hypoxic MM cells (mean fluorescent intensity shifts: Isotype=90, normoxic = 520, hypoxic = 1100). These results suggest that hypoxic MM cells will home to normoxic BM niches more efficiently than normoxic MM cells. Finally, testing the recovery of the hypoxic MM cells after incubation in normoxic conditions showed that the expression of CXCR4 was completely downregulated to levels found in normoxic cells after 6 hrs of exposure to normoxia. Moreover, incubation of hypoxic MM cells in normoxic BMSCs- media or SDF1 restored their expression of cadherins and their ability to adhere to BMSCs. CONCLUSION: In conclusion, we have showed that hypoxia inhibited the proliferation of MM cells and decreased their adhesion to BMSCs, as a first step of egress and metastasize to new normoxic BM niches. Disclosures: Anderson: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

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Targeting the Clonogenic Side Population of Multiple Myeloma by Immunomodulatory Drugs (IMiDs) in the Context of Stromal Cell Microenvironment: Pathophysiologic and Clinical Implications.

Blood ◽

10.1182/blood.v114.22.954.954 ◽

2009 ◽

Vol 114 (22) ◽

pp. 954-954

Author(s):

Jana Jakubikova ◽

David N. Cervi ◽

John F. Daley ◽

Danka Cholujova ◽

Melissa G. Ooi ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Board Of Directors ◽

Cell Lines ◽

Stromal Cells ◽

Research Funding ◽

Side Population ◽

Drug Resistant ◽

Advisory Committees ◽

New Strategies

Abstract 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.

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Metabolomic Profiling Identifies Mechanisms Regulating Hypoxia-Induced Drug Resistance In Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.121.121 ◽

2013 ◽

Vol 122 (21) ◽

pp. 121-121

Author(s):

Patricia Maiso ◽

Daisy Huynh ◽

Yosra Aljawai ◽

John M. Asara ◽

Antonio Sacco ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Drug Resistance ◽

Energy Metabolism ◽

High Risk ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Hypoxic Conditions

Abstract Background Multiple Myeloma (MM) is the second most prevalent hematological malignancy and remains incurable, with a median survival of 3-7 years. However, despite the success of the new treatments, most patients still succumb to their disease. In about 20-25% of high-risk patients, MM progresses rapidly and does not respond to conventional therapies leading to rapid extramedullary disease and demise of these patients. One such regulator of dissemination and drug resistance is the dynamic process of oxygen deprivation or hypoxia. A number of studies show that hypoxia promotes neo-angiogenesis, cancer progression, epithelial-mesenchymal transition (EMT), acquisition of metastasis potential and stem-cell features, as well as resistance to therapy by activating adaptive transcriptional programs. Targeting hypoxia, and the metabolic pathways regulated by hypoxia in the tumor cells, could lead to novel opportunities for cancer therapy. Rapidly proliferating hypoxic cancer cells undergo a “metabolic switch” to anaerobic glycolysis. This altered energy metabolism has been shown to be associated with activated oncogenes and mutant tumor suppressors, which are more prevalent in patients with high-risk MM. We aimed 1) to examine the role of HIF-1a and HIF-2ain regulating drug resistance in vitro and in vivo and 2) to identify specific hypoxia-regulated genes and regulators of energy metabolism leading drug resistance in MM. Methods The effect of hypoxia was analyzed in different MM cell lines (MM1S, RPMI8226, U266 and H929) in basal conditions and after the treatment with bortezomib, dexamethasone or melphalan. The cytotoxicity was analyzed by means of MTT assay. Apoptosis studies were performed by flow cytometry. Gene expression profile of MM1S cells treated with bortezomib was compared in normoxia vs hypoxia using D-chip and GSEA softwares. Genes with expression changes greater or lower than 2 fold in either direction were selected. HIF1A and HIF2A knockdowns were performed in MM1S using lentiviral vectors. For metabolite collection, samples were re-suspended using HPLC grade water for mass spectrometry and analyzed using a 5500 QTRAP hybrid triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu). A total of 254 endogenous water soluble metabolites were analyzed. Results We observed that MM cell lines were resistant to bortezomib and melphalan in hypoxic conditions (12 hours at 0.5% of oxygen levels) compared to normoxic conditions. At transcriptional and protein level. cells treated with bortezomib in hypoxic conditions affected a large number of genes/proteins involved in cell cycle such us p21, p53 and p57, cell death and glucose metabolism. However, cell cycle arrest was not responsible for the resistance of MM cells to bortezomib that was observed in hypoxic conditions. Therefore, we investigated mechanisms that are mediated by hypoxia and can regulate drug resistance. HIF1A knockdown restored the effect of bortezomib in MM1S and increased the percentage of apoptosis in cells treated with bortezomib under hypoxic conditions. To further explore the role of hypoxia in the regulation of tumor metabolism downstream of HIF1A, metabolomic studies were performed to characterize metabolic alterations following bortezomib treatment in hypoxic and normoxic conditions. This analysis revealed that hypoxic tumor cells treated with or without bortezomib show significant metabolic changes involving multiple pathways, the most significant of which are intermediates in glucose metabolism such us glucose-6-phosphate, fructose-6-phosphate, 3-phosphoglycerate and phosphoenolpyruvate. We also observed a decrease in measured tricarboxylic acid cycle (TCA) cycle intermediates (citrate, fumarate and malate) after hypoxia exposure and a significant increase of LDHA levels. We assessed the metabolic response to several drugs and shRNAs targeting different glycolytic enzymes (HK2, PFKBP3, PFKBP4 and LDHA). Of these, the most significant changes were observed with LDHA knockdown where these overcame resistance to bortezomib in hypoxic conditions. Conclusion Hypoxic conditions are essential for drug resistance and glucose utilization. These data provide new therapeutic targets and associated biomarkers for the treatment of Multiple Myeloma. Disclosures: Ghobrial: Onyx: Membership on an entity’s Board of Directors or advisory committees; BMS: Membership on an entity’s Board of Directors or advisory committees; BMS: Research Funding; Sanofi: Research Funding; Novartis: Membership on an entity’s Board of Directors or advisory committees.

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Iron Overload Diminishes the Effectiveness of the Innate Immune Response in Thalassemia Major: a Possible Mechanism for Increased Infection Risk.

Blood ◽

10.1182/blood.v114.22.4071.4071 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4071-4071

Author(s):

Patrick B Walter ◽

Paul R Harmatz ◽

Annie Higa ◽

David Killilea ◽

Nancy Sweeters ◽

...

Keyword(s):

Flow Cytometry ◽

Immune Response ◽

Iron Overload ◽

Board Of Directors ◽

Innate Immune Response ◽

Research Funding ◽

Innate Immune ◽

Healthy Controls ◽

Advisory Committees ◽

Fluorescent Intensity

Abstract Abstract 4071 Poster Board III-1006 Introduction Infection is the second most common cause of death in thalassemia. The innate immune system provides a first line of defense against infection and specificity depends on pattern recognition receptors (PRRs) specific to microbial pathogens. One class of PRR called the toll-like receptors (TLRs) are important for transducing the signal for bacterial Lipopolysaccharide (LPS), resulting not only in cytokine production, but also in the control of extracellular iron levels through production of neutrophil gelatinase associated Lipocalin (NGAL). However, the exact role that NGAL plays and the expression level of PRRs are unknown in thalassemia. Thus, the goal in these studies is to investigate the relationship of iron overload to the innate immune cell expression of PRRs and NGAL in thalassemia. Patients and Methods Fifteen transfusion dependent thalassemia patients (11 – 29 yrs old) participating in the combination trial of deferasirox (an oral iron chelator) and deferoxamine were enrolled (Novartis sponsored CICL670AUS24T). Fasting blood samples were obtained i) at baseline after a 72 hr washout of chelator, and ii) at 6 and 12 months on study. Five healthy controls (13 - 18 yrs old) were also enrolled. Fresh monocytes were isolated using antibody-linked magnetic microbeads (Miltenyi Biotec Inc). Highly enriched populations of CD14+ monocytes were verified by flow cytometry. The expression of TLR4, also examined by flow cytometry is reported as the mean fluorescent intensity (MFI). In patients with thalassemia, liver iron concentration (LIC) was analyzed by biomagnetic susceptibility (“SQUID”, Ferritometer®). The plasma levels of NGAL were analyzed by ELISA. Results At baseline the expression of monocyte TLR4 (mean 18.8 ± 3.5 MFI) was reduced 30% compared to the healthy controls (mean 26.9 ± 7.6 MFI, p<0.05). The expression of TLR4 over the follow-up period of 52 weeks in patients receiving intensive combination chelator therapy significantly increased 27% / year (7 MFI / year, p=0.005). Interestingly the expression of monocyte TLR4 was negatively correlated with LIC (r=-0.6, p=0.04). Finally, thalassemia patients at baseline have significantly higher levels of NGAL (80 ± 20 ng/ml) compared to controls (42 ± 15 ng/ml, p=0.01). Conclusions These preliminary studies support the hypothesis that iron burden has a negative impact on the innate immune response in thalassemia as demonstrated by the decreased expression of TLR4. After intensive chelation, the levels of TLR4 increased, indicating that decreased iron overload with chelation may improve innate immune responsiveness. Finally, the iron transport protein NGAL is significantly elevated in thalassemia possibly acting to prevent essential iron uptake by pathogenic bacteria. Disclosures: Harmatz: Novartis: Research Funding; Apotex : Membership on an entity's Board of Directors or advisory committees; Ferrokin: Membership on an entity's Board of Directors or advisory committees. Vichinsky:Novartis: Consultancy, Research Funding.

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CYC065, a Potent Derivative of Seliciclib Is Active In Multiple Myeloma In Preclinical Studies

Blood ◽

10.1182/blood.v116.21.2999.2999 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2999-2999 ◽

Cited By ~ 1

Author(s):

Samantha Pozzi ◽

Diana Cirstea ◽

Loredana Santo ◽

Doris M Nabikejje ◽

Kishan Patel ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Preliminary Data ◽

Research Funding ◽

Preclinical Studies ◽

Dependent Manner ◽

Advisory Committees

Abstract Abstract 2999 Multiple myeloma (MM) is a treatable but incurable hematological malignancy and novel targeted therapies are under investigation. MM is characterized by dysregulation of the cell cycle, consequent to the overexpression of cyclins and their related kinases, the cyclins dependent kinases (CDK), a group of Ser/Thr proteine kinases. CDKs represent a promising therapeutic target, and inhibitors have been developed for anticancer treatment. We have previously studied seliciclib in the context of MM. CYC065, a second generation CDK inhibitor is the more potent derivative of seliciclib. It is mainly active on CDK 2, 5 and 9, involved in progression of the cell cycle and protein transcription. It has already shown promising results in preclinical studies in breast cancer and acute leukemia. We tested CYC065 in in vitro experiments in MM. Our preliminary data in 7 MM cell lines showed cytotoxicity of CYC065, both in MM cell lines sensitive as well as resistant to conventional chemotherapy, with an IC50 ranging between 0.06 and 2μ M, at 24 and 48h. Tritiated thymidine uptake assay confirmed the antiproliferative effects of CYC065 in MM, and its ability to overcome the growth advantage conferred by co-culture with bone marrow stromal cells derived from MM patients, and cytokines like interleukin 6 (10ng/ml) and insulin like growth factor-1 (50ng/ml). The anti-proliferative effect was evident both at 24 and 48h, starting at concentrations as low as 0.015μ M. The AnnexinV/PI assay in the MM1.s cell line confirmed CYC065's ability to induce apoptosis in a time dependent manner starting at 9 hours of treatment, at a concentration of 0.125 μ M, inducing 82% of apoptosis after 48h of exposure. Cell cycle analysis in the same MM1.s cell line showed an increase of subG1 phase, starting at 9 hours of treatment, at 0.125 μ M of CYC065. Preliminary results of western blot analysis confirmed the apoptotic effect of CYC065 in the MM1s cell line, highlighted by the cleavage of caspase 3, 8, 9 and PARP. The compound was tested in primary CD138+ cells isolated from three refractory MM patients, confirming its efficacy at 0.125 μ M, both at 24 and 48h. Comparative analysis in PBMCs from normal donors, for the evaluation of the drug toxicity is ongoing and will be presented. In conclusion our preliminary data confirm the efficacy of CYC065 in MM cell lines and primary MM cells, at nanomolar concentrations. Ongoing mechanistic and in vivo studies will delineate its role in the now increasing spectrum of CDK inhibitors in MM and better define its potential for clinical development in MM. Disclosures: Green: Cyclacel: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Acetylon: Research Funding.

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Tris DBA Palladium Overcomes Hypoxia Mediated Drug Resistance in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.2978.2978 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2978-2978

Author(s):

Pilar De La Puente ◽

Barbara Muz ◽

Feda Azab ◽

Micah John Luderer ◽

Jack L. Arbiser ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Proliferation ◽

Drug Resistance ◽

Cell Lines ◽

Research Funding ◽

Caspase 3 ◽

Cellular Processes ◽

Hypoxic Conditions

Abstract Introduction: Despite recent progress in novel and targeted therapies, multiple myeloma (MM) remains a therapeutically challenging incurable disease. The regulation of important cellular processes and its link to cancer presented Src as an attractive target for MM. Src is a non-receptor protein tyrosine kinase which regulates multiple fundamental cellular processes including cell growth, migration, survival and differentiation. Activated Src in cancer lead to studies with Src as a target for anti-cancer drugs, and numerous Src inhibitors have become available to test the importance of Src in tumor initiation and progression. In MM, it has been described that in cell lines and MM patient-derived tumors, c-Src is constitutively activated, which plays an important role in drug resistance mechanisms. Tris dibenzylideneacetone dipalladium (Tris DBA), a small-molecule palladium complex, was shown to reduce Src/NMT-1 complex in melanoma cells, as well as inhibit downstream signaling including mitogen-activated protein kinase (MAPK kinase) and phosphoinositol-3-kinase (PI3K). We suggest a novel strategy to improve the treatment of MM and overcome the drug resistance for the current therapeutic agents by specific inhibition of Src in MM cells by an organopalladium compound, Tris DBA. Methods: Tris DBA was prepared by Dr. Arbiser. MM cell lines (MM.1S, MM.1R, H929, RPMI-8826, and OPM2) and PBMCs were cultured with Tris DBA (0-10 µM) for 24h. MM cells were analyzed for cell proliferation by MTT assay; cell cycle by DNA staining with PI and analyzed by flow cytometry; apoptosis was analyzed by Annexin V/PI staining and analyzed by flow cytometry; and cell signaling associated with proliferation, cell cycle, and apoptosis was analyzed by western blotting. In addition, cell proliferation assay of Tris DBA with or without combination of proteasome inhibitors (PIs) bortezomib or carfilzomib for 24h was analyzed on the proliferation of MM cells in normoxic or hypoxic conditions. Moreover, we tested the effect of combination treatment on cell cycle and apoptosis signaling under normoxic conditions. We then evaluated the effect of Tris DBA on HIF1α expression, migration and drug resistance under normoxic or hypoxic conditions. Results: The Src inhibitor Tris DBA reduced the proliferation of MM cell lines with an IC50 of about 1.5 - 3 µM after 24h treatment as a single agent, while none of the normal PBMC controls showed effect on their proliferation in the same dose range. These results were consistent with the decreased expression of proliferation signaling proteins from MAPK pathways (pERK), as well as PI3K (pS6R). Src inhibition led to the induction of a sub-G1 peak, which indicated accumulating apoptotic cells shown by DNA staining with PI. Apoptosis was then analyzed by Annexin/PI and confirmed by cleavage of caspase-3 and PARP. We found that Tris DBA synergized with bortezomib and carfilzomib by inhibiting proliferation of MM cells and reducing cell cycle protein signaling more than either of the drugs alone. Moreover, the Tris DBA/Bortezomib or Tris DBA/Carfilzomib combination therapies significantly increased apoptosis by caspase-3 cleavage more than treatment with either proteasome inhibitor individually. Tris DBA inhibited HIF1α expression in both normoxic and hypoxic conditions. HIF1α is an important target for hypoxia-driven drug resistance. Our studies confirmed hypoxia promoted faster chemotaxis of MM cells towards the chemo-attractants found in stromal cell conditioned media, and that Tris DBA treatment could overcome this hypoxia-induced effect. In addition, the development of hypoxia-induced drug resistance to individual bortezomib or carfilzomib treatment was overcome with combination treatment of Tris DBA under hypoxic conditions. Conclusions: Tris DBA reduces proliferation and induces G1 arrest and apoptosis in MM cells. Tris DBA synergized with PIs reducing proliferation and cell cycle signaling, as well as increasing apoptosis more than each drug alone. Tris DBA overcame hypoxia-induced effects such as enhanced chemotaxis or drug resistance to PIs by inhibition of HIF1α expression. Moreover, we found that Tris DBA is an effective anti-myeloma agent alone or in combination with other targeted drugs and that it reverses hypoxia-induced drug resistance in myeloma. These results suggest the use of Tris DBA as a new therapeutic agent in relapsed refractory myeloma. Disclosures Arbiser: ABBY Therapeutics: Other: Jack L Arbiser is listed as inventor on a US Patent for imipramine blue. He is cofounder of ABBY Therapeutics, which has licensed imipramine blue from Emory University.. Azab:Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding.

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Single-Cell Pathway Enrichment and Regulatory Profiling of Multiple Myeloma across Disease Stages

Blood ◽

10.1182/blood-2019-131361 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 364-364

Author(s):

Tianjiao Wang ◽

Hua Sun ◽

Daniel Cui Zhou ◽

Ruiyang Liu ◽

Lijun Yao ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Cell Types ◽

Differentially Expressed ◽

P Value ◽

Advisory Committees ◽

Pathway Enrichment

Multiple myeloma (MM) is a hematological malignancy, defined by aberrant monoclonal proliferation of plasma cells in the bone marrow, that to date remains an incurable disease despite advances in treatment. Key genetic and epigenetic alterations that drive MM pathogenesis have been identified, but a comprehensive profile of affected cellular pathways has yet to be fully characterized. In this study, we integrate whole-genome and whole-exome sequencing data with single-cell RNA sequencing (scRNA-seq) data from 13 patients across multiple treatment stages to 1) assess differential pathway enrichment between tumor subpopulations, 2) trace the clonal evolution of dominant disease mechanisms, and 3) investigate signaling interactions between surrounding cell types. We also analyzed bulk genomic and transcriptomic data from 662 additional Multiple Myeloma Research Foundation (MMRF) tumor samples as a large reference cohort for highly prevalent pathway disturbances. To assess whether tumor subpopulations rely on different oncogenic programs for proliferation, we analyzed the differential expression of key genes (FDR-adjusted p-value <0.05) in 12 canonical oncogenic pathways. Cell cycle, Hippo, RTK/RAS, and NFkB pathways contain the highest numbers of differentially expressed genes, with certain subclusters upregulating as many as 25% of annotated cell cycle genes and over 90% of annotated Hippo genes, whereas p53, Notch, Nrf2, and DNA repair genes tend to be uniformly expressed across subpopulations. Next, we evaluated changes in pathway enrichment across different disease timepoints, with the goal of capturing the reorganization of functional profiles through successive treatment and relapse cycles. We assessed statistical enrichment of pathways containing differentially expressed genes (DEGs) unique to Smoldering Multiple Myeloma (SMM), primary, and relapse stages using the KEGG pathway database (n = 2, 17, and 7 pathways, respectively; FDR-adjusted p-value of enrichment < 0.05). SMM is the only stage where hematopoietic differentiation and the PI3K-Akt pathways are variably expressed between plasma cell subpopulations, suggesting that these pathways may play a role in initiating events. Only primary tumor samples show significant intra-tumor variability of p53 regulation, which is lost in the relapsed tumor and may reflect selection due to treatment. Relative to SMM, primary and relapse samples are enriched for changes in the MAPK, NFkB, RAP1, and cell cycle pathways, indicating potential sources of tumor resistance. We then analyzed pathway enrichment within the tumor microenvironment to enhance our understanding of tumor development in the context of surrounding tissues. We see frequent changes in many immune cell types in TLR signaling as the disease progresses, driven by differential expression of NFkB1A, JUN, and FOS, all of which are key upstream regulators of the AP-1 pathway and responders to the MAPK and PI3K signaling cascades. These microenvironment changes may be complementary to the PI3K and MAPK dysregulation observed in tumor plasma cells. Proteasome and ubiquitin genes, which affect secretion, autophagy, and apoptosis pathways that may be relevant to MM pathogenesis are also frequently differentially expressed in immune cells between disease stages. Finally, we integrate bulk whole-exome and whole-genome sequencing analysis (from both the 13-patient cohort and MMRF) to obtain a more complete understanding of how pathways become dysregulated in MM. Our findings advance the understanding of how MM tumor subpopulations differentially regulate cellular pathways and interact within the tumor microenvironment. Disclosures O'Neal: Wugen: Patents & Royalties: Patent Pending; WashU: Patents & Royalties: Patent Pending. Rettig:WashU: Patents & Royalties: Patent Application 16/401,950. Oh:Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Vij:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Honoraria; Karyopharm: Honoraria; Sanofi: Honoraria; Takeda: Honoraria, Research Funding. DiPersio:Amphivena Therapeutics: Consultancy, Research Funding; Magenta Therapeutics: Equity Ownership; Karyopharm Therapeutics: Consultancy; Incyte: Consultancy, Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; NeoImmune Tech: Research Funding; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees.

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Methyljasmonate, An Inhibitor of Glycolytic Energy Production, Displays Pre-Clinical Activity against Multiple Myeloma Cells.

Blood ◽

10.1182/blood.v114.22.1741.1741 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1741-1741

Author(s):

Steffen Klippel ◽

Jana Jakubikova ◽

Jake Delmore ◽

Melissa G. Ooi ◽

Douglas McMillin ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Death ◽

Cancer Cells ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Energy Production ◽

Advisory Committees ◽

Normal Cells

Abstract 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.

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Role of Selectins in the Pathogenesis of Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.951.951 ◽

2009 ◽

Vol 114 (22) ◽

pp. 951-951 ◽

Cited By ~ 1

Author(s):

Abdel Kareem Azab ◽

Phong Quang ◽

Feda Azab ◽

Costas M Pitsillides ◽

John T Patton ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Endothelial Cells ◽

Cell Adhesion ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Advisory Committees

Abstract Abstract 951 INTRODUCTION: Multiple Myeloma (MM) is characterized by widespread disease at diagnosis with the presence of multiple lytic lesions and disseminated involvement of the bone marrow (BM), implying that the progression of MM involves a continuous re-circulation of the MM cells in the peripheral blood and re-entrance into the BM. Selectins are adhesion molecules expressed by activated endothelium of venules and leukocytes, and are involved in the primary interaction of lymphocytes with the endothelium of blood vessels. The binding of selectins serves as a biologic brake, making leukocyte quickly decelerate by rolling on endothelial cells, as the first step of extravasation. In this study, we have investigated the role of selectins and their ligands in the regulation of homing of MM Cells to the BM and the therapeutic implications of this role. METHODS AND RESULTS: We have used flow cytometry to characterize the expression of E, L and P-selectins and their ligands on MM cell lines, patient samples and on plasma cells from normal subjects. We found that all MM cell lines and patient samples showed high expression of L and P, but little of no E-selectin. While normal plasma cells showed low expression of all selectins and ligands.(give numbers) A pan-selectin inhibitor GMI-1070 (GlycoMimetics Inc., Gaithersburg, MD) inhibited the interaction of recombinant selectins with the selectin-ligands on the MM cells in a dose response manner. We have tested the role of the selectins and their ligands on the adhesion of MM cells to endothelial cells and found that MM cells adhered preferentially to endothelial cells expressing P-selectin compared to control endothelial cells and endothelial cells expressing E-selectin (p<0.05). Moreover, we found that blockade of P-selectin on endothelial cells reduced their interaction with MM cells (p<0.01), while blockade of E and L-selectin did not show any effect. Treating endothelial cells with GMI-1070 mimicked the effect of blocking P-selectin. Moreover, we found that treating endothelial cells with the chemokine stroma cell-derived factor-1-alpha (SDF1) increased their expression of P but not E or L-selectin detected by flow cytometry. Neither the blockade of each of the selectins and their ligands nor the GMI-1070 inhibited the trans-well chemotaxis of MM cells towards SDF1-alpha. However, blockade of P-selectin (p<0.001) on endothelial cells by GMI-1070 inhibited the trans-endothelial chemotaxis of MM cells towards SDF1-alpha. Both adhesion to endothelial cells and activation with recombinant P-selectin induced phosphorylation of cell adhesion related molecules including FAK, SRC, Cadherins, Cofilin, AKT and GSK3. GMI-1070 decreased the activation of cell adhesion molecules induced by both recombinant P-selectin and endothelial cells. Using in vivo flow cytometry we found that both anti P-selectin antibody and GMI-1070 prevented the extravasation of MM cells out of blood vessels into the bone marrow in mice. Moreover, we found that, in a co-culture system, endothelial cells protected MM cells from bortezomib induced apoptosis, an effect which was reversed by using GMI-1070, showing synergistic effect with bortezomib. CONCLUSION: In summary, we showed that P-selectin ligand is highly expressed in MM cells compared to normal plasma cells, and that it plays a major role in homing of MM cells to the BM, an effect which was inhibited by the pan-selectin inhibitor GMI-1070. This provides a basis for testing the effect of selectin inhibition on tumor initiation and tumor response to therapeutic agents such as bortezomib. Moreover, it provides a basis for future clinical trials for prevention of MM metastasis and increasing efficacy of existing therapies by using selectin inhibitors for the treatment of myeloma. Disclosures: Patton: GlycoMimetics, Inc: Employment. Smith:GlycoMimetics, Inc: Employment. Sarkar:GlycoMimetics, Inc: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Magnani:GlycoMimetics, Inc.: Employment. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

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Proteasome Inhibitor Treatment in Multiple Myeloma Can Mobilize Hematopoietic Stem Cells in the Absence of G-CSF

Blood ◽

10.1182/blood.v124.21.2452.2452 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2452-2452

Author(s):

John N. Allan ◽

David Jayabalan ◽

Ruben Niesvizky ◽

Tomer M Mark ◽

Roger Pearse ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Stem Cell ◽

Board Of Directors ◽

Proteasome Inhibitor ◽

Research Funding ◽

Fold Change ◽

Multiparameter Flow Cytometry ◽

Advisory Committees ◽

Hematopoietic Stem

Abstract Introduction Proteasome inhibitor (PI) use in patients (pts) with multiple myeloma (MM) has been associated with increased hematopoietic stem/progenitor cell (HSPC) collection yields in both induction and autologous stem cell collection settings (Niesvizky et al., 2013). Animal models have confirmed this observation (Ghobadi et al., 2012). The mechanism remains unclear, but there is suggestion PI treatment affects pathways associated with HSPC anchoring and migration (Niesvizky et al., 2013). The effect of PIs on HSPC migration in the absence of filgastrim (G-CSF) stimulation remains unknown. We sought to characterize the molecular mechanisms of HSPC mobilization in a cohort of pts undergoing active PI treatment. Methods MM pts undergoing treatment with PIs were consented to obtain peripheral blood (PB) under IRB approval. Pts were eligible if they had symptomatic MM and were undergoing treatment with a PI. Pts receiving alkylating chemotherapy (such as cyclophosphamide) in combination with a PI were excluded. Pts were enrolled on the first day of a new cycle containing a PI. PB was drawn prior to administration of the PI (T0) and just prior to the next dose of PI, 24 or 72 hours later (T1), depending on whether the pt was receiving carfilzomib or bortezomib, respectively. PB mononuclear cells were collected and purified with Ficoll-Paque, viably frozen in CS-10 freezing medium and stored in liquid nitrogen. Serum samples were collected after a 1:2 dilution with PBS and stored at -80oF. Cells were later thawed to perform multiparameter flow cytometry and colony forming unit (CFU) assays. Multiparameter flow cytometry was performed using a BD LSR-II and analyzed using FloJo V9.0 software. Cells were gated on CD45dim SSC-lo characteristics. HSPCs were defined as CD34+/CD133+. Pts were stratified into 3 groups (>2, 1-2, <1) based on fold change in peripheral HSPCs from baseline T0. Expression of surface markers including CD38, CD184, CD202b, CD25, CD90 and CD31 within the HSPC population, were analyzed. Serum protein concentrations were analyzed using ELISAs. Results Twenty-three pts consented and collected at the 2 prespecified time points. Six pts (26%) increased the percentage of peripheral HSPCs>2 fold. Nine (39%) and 8 (35%) pts increased the percentage of HSPCs 1-2 fold and <1 fold over T0 percentage, respectively. There were no statistical differences within the 3 groups, in baseline characteristics, prior chemotherapy, use of IMIDs, or radiation exposure history. There was a significant positive correlation between peripheral HSPC fold change and CFU formation p=0.003 indicating the mobilized HSPC population’s capacity to form progeny. Furthermore, there was a significant negative correlation between fold change of HSPCs and CD90 expression on CD34+ CD133+ CD38- stem cell populations at T1 p=0.032. To determine changes in serum proteins as a result of PI treatment that could contribute to HSPC mobilization we evaluated TGF-ß levels in 13 pt plasma samples. Two pts from the>2fold group were available and revealed TGF-ß levels increase 67.24 pg/mL compared to a decrease of 17.67 pg/mL in 5 pts in the <1fold group trending towards significance p=0.094. Baseline levels of TGF-ß in the two groups,>2fold and <1fold were 18.1 pg/mL and 30.1 pg/mL respectively, which was not significant. Discussion Observations have noted increased HSPC yields in animal models and MM pts after treatment with PIs in both induction and mobilizing regimens (Ghobadi et al., 2012; Niesvizky et al., 2013). Here we demonstrate that treatment with PIs is associated with increases in peripheral HSPC percentages in approximately 2/3 of MM pts despite the lack of concurrent G-CSF. Decreased CD90 has previously been observed in peripherally mobilized HSPC products and, similar to TGF-ß, plays a role in regulation of Rhokinase GTPase pathways known to affect migration and adherence of many different cell types (Tsuchiya et al., 1997; Kim et al., 2006; Wen et al., 2013; Kim et al., 2014). Our study shows a correlation between decreased CD90 expression and fold increase of peripheral HSPCs. We also found an increase in TGF-ß serum levels after treatment in the>2fold group compared to the <1fold group, which may approach statistical significance with more sampling. These findings may help understand the failure to collect adequate HSPCs in a subset of MM pts and could highlight new pathways to disrupt and improve HSPC mobilization regimens. Disclosures Niesvizky: Onyx Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding, Speakers Bureau; Millennium: The Takeda Oncology Company: Consultancy, Research Funding, Speakers Bureau. Mark:Onyx: Research Funding, Speakers Bureau; Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Rossi:Celgene: Speakers Bureau.

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Ex Vivo Assessment of Tnb-383B, a Bcma-Bispecific Antibody, Against Primary Tumor and Endogenous T Cells from Relapsing Multiple Myeloma Patients

Blood ◽

10.1182/blood-2018-99-118881 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1940-1940 ◽

Cited By ~ 2

Author(s):

David M. Foureau ◽

Manisha Bhutani ◽

Myra Robinson ◽

Fei Guo ◽

Duy Pham ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

T Cells ◽

Board Of Directors ◽

Research Funding ◽

Ex Vivo ◽

Negative Control ◽

Advisory Committees ◽

Measured Variable ◽

Dose Dependent

Abstract INTRODUCTION: The human B-Cell maturation antigen (BCMA) is a surface marker that is highly expressed on plasma cells and has been recognized as a novel target in multiple myeloma (MM). TNB-383B is a fully human bispecific monoclonal IgG4 antibody. TNB-383B consists of 2 heavy and 1 light chain(s) paired through knob-in-hole technology. The first heavy chain and a kappa light chain form the paratope to recognize and bind human CD3. The second heavy chain is comprised of two identical VH domains in sequence and targets human BCMA with high affinity and avidity. Herein, we describe the ability of TNB-383B to mediate killing of patient-derived tumor cell lysis by endogenous T-cells was assessed ex vivo. METHODS: Bone marrow mononuclear cells (BMMCs) were isolated by density gradient centrifugation from 7 relapsed MM patients enrolled in an IRB-approved biospecimen collection protocol. Freshly isolated BMMC subsets were characterized by flow cytometry, specifically plasma cell (PC) / cytotoxic T cell (CTL) distribution, PC BCMA expression and PC viability were determined. BMMCs were then incubated for 24h (± 2h) with TNB-383B, or negative control, at concentrations ranging from 0.001-1μg. Following incubation, PC lysis, viability, BCMA expression, as well as CTL distribution and degranulation were assessed by flow cytometry. Tukey's sequential trend test was performed for each measured variable, utilizing ANOVA models and contrast statements, to detect linear dose response trends to TNB-383B or negative control treatments. Additionally, a parametric model (EMax) was used for each measured variable to estimate dose response curves, interpolating between tested doses. Two-way factorial ANOVA was utilized to compare the main effects of E:T ratio (or PC phenotype) and dose level and the interaction effect between E:T ratio and dose level on measured variables. RESULTS: Dose-dependent PC lysis was triggered by TNB-383B at concentrations as low as 0.001μg (p=0.0102) while no significant loss of PC was observed with negative control (Figure). This effect was coupled with significant CTL degranulation as expressed by increased CD107a mean fluorescence intensity (MFI) specific to TNB-383B treatment (p=0.0153 at 1μg). Although apoptotic rates (7-AAD+, Annexin V+) of the remaining PC tend to increase among TNB-383B treated compared with isotype control-treated cells, this trend was not significant. As opposed to CTL degranulation, CTL proliferation was not significantly triggered by TNB-383B but was significantly increased when BMMCs were exposed to negative control antibody (p=0.0057 at 0.001 μg). BMMC containing effector to target (E:T) ratio >10 contained lower viable (7-AAD-) PC and higher apoptotic PC counts compared with BMMC specimen with E:T ratio <10 (p<0.001). Using CD45 expression as a surrogate marker of PC maturation and BCMA expression, CD45+ PC displayed higher BCMA expression than CD45- PC (p=0.0189) and were more sensitive TNB-383B-induced killing (p<0.001). Noticeably, overall BCMA expression pre/post TNB-383B exposure remained unaltered. CONCLUSION: Taken together, our findings demonstrate TNB-383B triggers primary PC lysis and CTL degranulation in a dose-dependent fashion ex vivo. The ex vivo bispecific monoclonal antibody assay employed in this study allowed us to identify underlying biological drivers of PC killing efficacy by TNB-383B and may provide a valuable preclinical platform to screen bispecific antibodies and clinical platform to identify mechanism of primary or acquired resistance to the drug. Enrollment of patients with relapsed/refractory MM into a phase I clinical trial with TNB-383B is expected in early 2019. Figure. Figure. Disclosures Foureau: Teneobio Inc.: Research Funding. Pham:Teneobio Inc.: Employment. Force Aldred:Teneobio Inc.: Employment. Buelow:Teneobio Inc.: Employment. Voorhees:Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: served on an IRC; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Other: served on an IRC; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: served on an IRC; Amgen Inc.: Speakers Bureau; TeneoBio: Consultancy, Membership on an entity's Board of Directors or advisory committees. Usmani:Abbvie, Amgen, Celgene, Genmab, Merck, MundiPharma, Janssen, Seattle Genetics: Consultancy; Amgen, BMS, Celgene, Janssen, Merck, Pharmacyclics,Sanofi, Seattle Genetics, Takeda: Research Funding.

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