ALBERT

Description: SGN-40, a humanized immoglobulin G1 (IgG1) anti-CD40 monoclonal antibody, mediates cytotoxicity against human multiple myeloma (MM) cells via several mechanisms in vitro. These include induction of cytotoxic ligands of TNFR family and suppression of IL-6-induced proliferative and antiapoptotic effects, as well as antibody-dependent cell-mediated cytotoxicity (ADCC). Since 〉 80% of primary patient MM cells express CD40, targeting CD40 using SGN-40 presents a potential novel treament strategy, and a phase I clinical study of SGN-40 in patients with refractory or recurrent MM is ongoing. We recently reported that Thalidomide and immunomodulatory drugs (IMiDs) target both MM cells and the bone marrow (BM) microenvironment, and activate NK cells via induction of IL-2 production. In the present study, we therefore evaluated the effects of IMiD3 on the direct antiproliferative and apoptotic effects of SGN-40, as well as on ADCC against both MM cell lines and patient MM cells (CD40+CD138++). SGN-40 and IMiD3 induced synergistic growth inhibition, assayed by [3H] thymidine uptake, in dexamethasone (Dex)-sensitive MM.1S and Dex-resistant MM.1R lines, 2 other CD40-positive MM cell lines, as well as 2 patient MM cells. The temporal sequence of SGN-40 and IMiD3 treatment did not alter growth inhibition. The combination of SGN-40 and IMiD3 significantly increased MM apoptosis, evidenced by enhanced cleavage of caspase 3/8/PARP and increased subG0 cells compared with either single agent. The addition of IMiD3 to target cells and effector cells moderately increased specific lysis in any MM cell line, whereas pretreatment of target cells with IMiD3 significantly augmented sensitivity of all MM lines to ADCC and pretreatment of effector cells also improved specific MM cell lysis. In addition, preincubation of both effector and tumor cells with IMiD3 greatly enhanced specific lysis of MM cell lines and 2 patient MM cells in ADCC assay, associated with a significant increase 38+3% in natural killer cells (CD56+CD16+ and CD14-CD3-) following IMiD3 treatment. IMiD3 not only improved natural cytotoxicity of NK cells, but also significantly induced the CD56dimCD16+CD3- NK subset, which is a more potent mediator of ADCC against MM than the CD56bright NK subset. Moreover, IMiD3 treatment upregulates CD40L expression on CD56+CD3- NK effectors: IMiD3 (2 μM) induces CD40L upregulation equivalent to IL-2 (1000 unit/ml). Finally, combined SGN-40 and IMiD3 augments NK cell proliferation, which is associated with enhanced AKT/NF-kB and ERK activation. Taken together, our studies show that the addition of IMiD3 to SGN-40 results in synergistic cytotoxicity mediated via direct antiproliferative and apoptotic effects therefore increased sensitivity of MM cells to ADCC by inducing the cytotoxic NK subset. These studies establish the framework for the development of SGN-40 and IMiD3 in a new treament paradigm to both target MM cells directly and to induce immune effectors against MM.

Description: Cell membrane protein CS1 is highly expressed by tumor cells from the majority of multiple myeloma (MM) patients (〉95%) regardless of cytogenetic abnormalities and response to current treatments. Furthermore, CS1 is detected in MM patient sera and correlates with active MM. However, its role in MM pathophysiology is undefined. In the present study, we first generated CS1 null OPM2 MM cells using lentiviral CS1 short interfering RNA. Specific CS1 knockdown was confirmed by depletion of CS1 mRNA and membrane protein, whereas CS1 was expressed in parental OPM2 and OPM2 cells infected with control lentiviral vector (cntOPM2). Immunoblotting of phopho-site of multiple kinase screen analysis showed decreased phosphorylation of ERK1/2, AKT, and STAT3 in CS1null OPM2 cells vs. cntOPM2 cells. Serum deprivation markedly blocked survival at earlier time points in CS1null OPM2 cells vs. cntOPM2 cells. Earlier apoptosis in CS1null OPM2 cells correlated with earlier activation of caspases, PARP cleavage, and increased proapoptotic proteins BNIP3, BIK. CS1 knockdown further delayed development of OPM2 tumor and prolonged survival in mice. CS1null OPM2 cells failed to grow tumors in the majority of mice (n=8) at 5 weeks after cell inoculation, whereas cntOPM2 cells formed tumors within 1.5 weeks in all animals (n=8). Interestingly, CS1 was expressed in tumors that developed late in mice injected with CS1null OPM2 cells. Concomitantly, we overexpressed CS1 in CS1-low expressing U266 cells by transfecting an expressing plasmid pflagCS1 or control vector. Enforced CS1 expression enhanced U266 cell growth and survival. In contrast to the majority of U266 cells (〉95%) that grow in suspension in standard tissue culture flasks, all U266CS1 cells exhibited adherent growth and homotypic adhesion. Importantly, overexpressed CS1 increased adhesion of U266 and MM1S cells to BMSCs. Furthermore, U266CS1 cells formed more and larger colonies in methylcellulose than U266 cells. Interestingly, tumors that developed in mice injected with U266 cells expressed significantly higher levels of CS1 than injected U266 cells; moreover, exercised tumors grew in an adherent manner in vitro. Overlapping differentially expressed genes in U266CS1 vs. U266 and CS1null OPM2 vs. cntOPM2 was next analyzed by gene expression profiling. Importantly, c-maf pathway was significantly upregulated in U266CS1 vs. U266 cells and downregulated in CS1null OPM2 vs. cntOPM2 cells, as evidenced by differentially expressed c-maf and its target genes, i.e., cyclin D2, integrin αE/β7 at both mRNA and protein levels. Myeloma cell adhesion-induced VEGF secretion by BMSCs was greater with U266CS1 than U266 cells. Finally, immunoblotting showed upregulation of c-maf and cyclin D2 in U266 tumors overexpressing CS1. These studies provide direct evidence of the role of CS1 in myeloma pathogenesis, define molecular mechanisms regulating its effects, and further support novel therapies targeting CS1 in MM.

Description: Vascular endothelial growth factor (VEGF) induces proliferation of MM cells and induces interleukin-6 (IL-6) secretion in a paracrine loop involving MM cells and bone marrow stromal cells. In turn, IL-6 triggers multiple myeloma (MM) cell proliferation and also protects against apoptosis by upregulating Myeloid-cell-leukemia 1 (Mcl-1), a critical survival protein in MM cells. The goal of our study was to investigate the role of Mcl-1 in VEGF induced-proliferation and protection against apoptosis. Using two murine embryonic fibroblast cell lines as a model (a Mcl-1 deleted cell line and its wild type: Mcl-1Δ/null and Mcl-1wt/wt MEFs, respectively), we here demonstrate that deletion of Mcl-1 reduces fetal bovine serum (FBS), VEGF, and IL-6 induced-proliferation. In addition, we demonstrate that the percentage of cells in S phase is lower in Mcl-1Δ/null compared to Mcl-1wt/wt MEFs (21% (+/−1) versus 30% (+/− 3), respectively). Taken together, these results demonstrate that Mcl-1 is required to mediate VEGF, Il-6 and FBS-induced-proliferation and cell cycle progression. To highlight the key anti-apoptotic role of Mcl-1 in MM cells, humans MM1s cells were transfected with Mcl-1 siRNA. Specific inhibition of Mcl-1 was associated with decreased proliferation (42% and 61% decreases at 24 and 48 h, respectively) and induction of apoptosis (subG1 peak: 22% and 41% in Mcl-1 siRNA transfected cells versus 15% and 15 % in non-transfected cells at 24 and 48 h, respectively), confirming that Mcl-1 is critical for both proliferation and protection against apoptosis in MM cells. In 3 human MM cell lines (MM1s, U266 and MM1R) and MM patient cells we next showed that Mcl-1 protein expression, but not other bcl-2 family members, is upregulated by VEGF in a time and dose manner; and conversely that the pan-VEGF inhibitor GW654652, blocks VEGF induced-upregulation of Mcl-1. Furthermore using flow cytometry with a double staining (CD38-FITC and Apo 2.7-PE), we demonstrate that VEGF protects MM patient cells from FBS-starvation-induced-apoptosis: the percentage of apoptotic MM patient cells (CD38++ and Apo 2.7+) in non starved medium (RPMI 1640 supplemented with 10% FBS) was 15% versus 93% in starved medium (RPMI 1640 supplemented with FBS 2%), and 48% in starved medium supplemented with 25ng/ml VEGF. In conclusion, our study demonstrates that VEGF protects MM cells against apoptosis, and that VEGF-induced MM cell proliferation and survival is mediated via Mcl-1. these studies provide the preclinical framework for novel therapeutics targeting both Mcl-1 and/or VEGF to improve patient outcome in MM.

Description: Abstract 330 Background: Our previous study demonstrated that inhibition of nicotinamide phosphoribosyltransferase (Nampt) acts by severely depleting intracellular NAD+ content and thus eliciting mitochondrial dysfunction and autophagic MM cell death. The proteasome inhibitor Bortezomib induces anti-MM activity by affecting a variety of signaling pathways. However, as with other agents, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we demonstrate that combining Nampt inhibitor and bortezomb induces synergistic anti-MM cell death both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. Material and Methods: We utilized MM.1S, MM.1R, RPMI-8226, and U266 human MM cell lines, as well as purified tumor cells from patients relapsing after prior therapies. Cell viability and apoptosis assays were performed using Annexin V/PI staining. Intracellular NAD+ level and proteasome activity were quantified after 12, 24, and 48h exposure to single/combination drugs by specific assays. In vitro angiogenesis was assessed by Matrigel capillary-like tube structure formation assay. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, and tubulin. CB-17 SCID male mice (n = 28; 7 mice/EA group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum free RPMI-1640 medium. When tumors were measurable (3 weeks after MM cell injection), mice were treated for three weeks with vehicle alone, FK866 (30mg/kg 4 days weekly), Bortezomib (0.5 mg/kg twice weekly), or FK866 (30 mg/kg) plus Bortezomib (0.5 mg/kg). Statistical significance of differences observed in FK866, Bortezomib or combination-treated mice was determined using a Student t test. Isobologram analysis was performed using “CalcuSyn” software program. A combination index 〈 1.0 indicates synergism. Results/Discussion: Combining FK866 and Bortezomib induces synergistic anti-MM activity in vitro against MM cell lines (P

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

Description: Abstract 2920 Background: Nicotinamide adenine dinucleotide (NAD+) is a coenzyme crucially involved in several cellular functions, including energy metabolism, reactive oxygen species scavenging, DNA repair, and gene expression. Intracellular NAD+ stores are continuously replenished through pathways whose activity depends on the tissue and availability of substrates. Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme in the NAD+ salvage pathway from nicotinamide. During neoplastic transformation, Nampt is upregulated to compensate for increased metabolic demands. It promotes myeloid and lymphoid differentiation and increases specific cytokine production (TNF- α, IL-6 and VEGF). Importantly, cancer and leukaemia cells appear to be more sensitive to Nampt inhibitor drugs than normal cells. The reasons for this selectivity are not fully understood, but may include aberrant metabolic demands and increased reliance on NAD+-dependent enzymes. Promising results obtained with Nampt inhibitors (such as FK866) in preclinical cancer models suggest that Nampt activity represents an innovative therapeutic target for novel anticancer agents. Methods: A panel of eighteen different MM cell lines, both sensitive and resistant to conventional and novel anti-myeloma drugs, as well as patient MM cells were used in the study. The mechanism of action of FK866 was investigated by Annexin-V/propidium iodide staining, thymidine incorporation, Western-blotting, and with lentivirus-mediated shRNAs. For the autophagy assay, EGFP-LC3+ cells were treated with FK866 and the number of GFP-LC3 punctae was analyzed and quantified by fluorescence microscopy and flow cytometry, respectivley. Intracellular NAD+ content was measured using a biochemical assay. Angiogenesis was measured in vitro using Matrigel capillary-like tube structure formation assay. Results: To study the role of Nampt in MM cells, we performed a protein analysis of this enzyme in eighteen MM cell lines. Nampt is constitutively activated in all cell lines tested. Moreover, patient MM cells highly express this enzyme whereas normal cells lack this protein. Indeed, the Nampt inhibitor FK866 decreased MM cell line viability in a dose and time dependent manner, with an IC50 ranging from 3–30nM. Similar results were observed in patient MM cells. Importantly, FK866 did not inhibit viability of normal peripheral blood mononuclear cells. Tritiated thymidine uptake assay confirmed the antiproliferative effects of FK866 in MM cell lines and patient cells. To examine the mechansim of action, we showed that intracellular NAD+ levels decreased with FK866 treatment at 24 and 48 hours. Furthermore, knock-down of Nampt by small interfering RNAs caused significant inhibition of MM cell growth. FK866 triggered anti-MM activity in our models of MM in the bone marrow (BM) microenvironment, confirming its ability to overcome the proliferative advantage conferred by the BM milieu. FK866 treatment also inhibited angiogenesis via suppression of pivotal MM pathways PI3K/AKT and ERK. In further studies to delineate its mechanisms of action, no activation of apoptosis was observed in treated-cells. Instead FK866 treatment resulted in a marked increase in autophagy, evidenced by autophagic vacuoles in the cytoplasm and proteolitic processing of endogenous LC3-I to LC3-II. FK866 inhibited mTOR signaling and triggered increased formation of EGFP-LC3 punctae, confirming involvement of autophagic cell death. Finally, combined FK866 with bortezomib (CI 〈 0.6), melphalan (CI 〈 0.9), and dexamethasone (CI 〈 0.8), induced synergistic cytotoxicity against MM cells. Conclusion: Our data therefore show a pivotal role of Nampt in MM cell growth, survival and drug resistance. The ability of FK866 to inhibit Nampt activity strongly supports its clinical evaluation to improve patient outcome in MM. Disclosures: Hideshima: Acetylon: Consultancy. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.

Description: Background: Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM), and lymphadenopathy in 20% of the patients, implying continuous trafficking of WM cells into and out of the BM and lymph nodes. The normal process of B-cell homing is regulated by cytokines, chemokines, and adhesion molecules. One of the most extensively studied chemokines in migration is stromal derived factor SDF-1 and its receptor CXCR4. Here we study the role of chemokine receptors, and the SDF-1/CXCR4 axis on migration and adhesion in WM. Methods: Flow cytometry for CXC and CC chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR2, CCR4, CCR5, CCR6 and CCR7), and adhesion molecules (VLA-4 and LFA-1) on WM cell lines (BCWM.1 and WM-WSU) and patient samples was performed. Migration was determined using the transwell migration assay (Costar, NY). Cells were placed in the upper chambers of the migration assay with 1% FCS medium in the presence of serial concentrations of SDF-1 in the lower chambers. After 4 hours of incubation, cells that migrated to the lower chambers were counted. Similarly, adhesion was determined using an adhesion assay (EMD Biosciences, San Diego, CA) with 96-well plated coated with fibronectin. Immunoblotting for proteins downstream of CXCR4 was performed. The CXCR4 inhibitor AMD3100 (10–100uM, Sigma, MO) and Gi protein inhibitor pertussis toxin PTX (10–200ng/ml, Sigma, MO) were used to inhibit CXCR4 signaling. Results: The following chemokine receptors were expressed on patient CD19+WM cells with over 30% expression: CXCR1 (mean 60%), CXCR2 (mean 47%), CXCR4 (mean 47%), CXCR5 (mean 69%), CCR4 (mean 54%) and CCR6 (mean 61%). Similar expression was observed on WM cell lines. We next determined the effect of SDF-1 on migration and signaling pathways in WM. SDF-1 (10–100nM) induced migration in a bell-shaped curve with 30nM inducing maximum migration (110% compared to control). SDF-1 30nM induced a rapid activation of signaling pathways downstream of CXCR4 including pERK1/2, pAKT, and pPKC at 1 min, with maximum activation at 5min. The CXCR4 inhibitor AMD3100 inhibited migration of BCWM.1 in the presence of 30nM SDF-1, with AMD3100 10uM inhibiting migration at 59% of control, and 20 to 50uM leading to a plateau in inhibition of migration at 54% of control. AMD3100 inhibited pERK and pPKC activation, downstream of CXCR4 in a dose-dependent fashion. Similar results were observed using PTX, with inhibition of migration of WM cells at 50% compared to control. To determine the role of SDF-1 on adhesion, we first demonstrated that WM cells from patients and cell lines expressed high levels of surface VLA-4 expression (mean 95% surface expression). WM cells had an increase in adhesion to fibronectin (VLA-4 ligand) compared to BSA control. AMD3100 10uM inhibited adhesion to fibronectin (63 % of control), indicating that the SDF-1/CXCR4 axis regulates adhesion. Conclusion: CXCR4 is highly expressed on WM cells and regulates migration and adhesion, indicating a potential role in regulating WM trafficking into the BM and lymph nodes. These studies provide the preclinical framework to study CXCR4 inhibitors in the regulation of homing and adhesion in WM.

Description: Background: Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation and accumulation of malignant plasma cells in the bone marrow and development of osteolytic bone lesions. Despite recent advances in treatment using novel therapeutics, MM remains incurable with high mortality rates. We have demonstrated in preclinical studies that CD8+ cytotoxic T lymphocytes (CTL) generated with immunogenic HLA-A2 or HLA-A24 peptides targeting XBP1(X-box binding protein 1), CD138 (Syndecan-1) and CS1 (SLAMF7) antigens induces robust cytotoxic activities against MM. The Phase 1/2a trials were completed or are in progress in the patients with smoldering multiple myeloma or triple negative breast cancer, respectively, using the HLA-A2 XBP1/CD138/CS1 multipeptide vaccine. The clinical data demonstrated that the multipeptide vaccine is safe and induces the XBP1/CD138/CS1-specific immune responses, evidenced by expansion of peptides-specific Tetramer+/CD45RO+memory CTL and Th-1 specific immune responses. Moreover, clinical trials combining with Lenalidomide or checkpoint inhibitor enhanced the CD8+ CTL activities induced by multipeptide vaccine, indicating the benefit of combination therapy. To expand the breadth and extent of the antigens-specific immunotherapy beyond XBP1/CD138/CS1, we have recently identified additional tumor-associated antigens (TAA) on tumor cells obtained from newly diagnosed MM patients (N=616). Here, we introduce a novel heteroclitic peptides specific to BCMA, the receptor forbinding of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). Due to its restricted expression pattern on MM cells and plasma cells along with its critical role in promoting MM cell growth, survival and drug resistance, we are currently in development of novel immunotherapeutic to target BCMA on MM cells with different therapeutic approaches. Objective: The aims of current study were to target BCMA as a TAA by generating the antigen-specific memory CD8+ CTL to induce effective and long-lasting immune response against MM. Findings: We report on immunogenic HLA-A2-restricted peptides derived from BCMA, which are capable of evoking antigen-specific immune responses against MM. The heteroclitic BCMA peptides displayed improved binding affinity/stability to HLA-A2 molecules from their native BCMA peptides. To define immunogenicity of the selected peptides, we generated BCMA-specific CTL (BCMA-CTL) by repeated stimulationof CD3+ T cells with respective heteroclitic peptide. The BCMA-specific engineered peptides evoked the expansion of antigen-specific CD8+ CTL and generated BCMA-CTL displaying high T cell activation (CD38, CD69) and co-stimulatory (CD40L, OX40, GITR) molecules expression. Additionally, a gradual expansion was observed in BCMA-specific memory CD8+ T cells, with a corresponding decrease in naïve CD8+ T cells. The BCMA-CTL demonstrated robust poly-functional immune responses with Th1-specific anti-MM activities [high IFN-g/IL-2/TNF-aproduction, CD8+ T cells proliferation, cytotoxicity] in antigen-specific and HLA-A2-restricted manner. The functional activities were directly correlated with the expansion of central memory CD8+ CTL in the BCMA-CTL generated from different HLA-A2+ individuals (Donor 1 BCMA-CTL: 81.0%, Donor 2 BCMA-CTL: 82.6%, Donor 3 BCMA-CTL: 67.0%). Finally, the combination with checkpoint inhibitor (anti-LAG3) or immune agonist (anti-OX40) enhanced the anti-tumor activities of BCMA-CTL, along with the induction of cytotoxic activities by central memory CD8+ T cell subset. Therefore, these studies suggest that heteroclitic BCMA peptides offer a therapeutic potential to effectively generate BCMA-specific CD8+ CTL targeting MM. Significance: Here, we introduce novel immunogenic engineered heteroclitic BCMA peptides capable of inducing antigen-specific memory CD8+ CTL with robust poly-functional immune responses against MM. These results provide the framework for therapeutic application of heteroclitic BCMA peptides in MM patients. They further support combination treatment options incorporating BCMA peptides-specific vaccine or BCMA peptides-specific adoptive T cells immunotherapy with anti-LAG3 and/or anti-OX40for patients with myeloma or other diseases expressing BCMA. Disclosures Munshi: OncoPep: Other: Board of director. Anderson:Celgene: Consultancy; Takeda Millennium: Consultancy; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership.

Description: Whether Kaposi’s sarcoma herpesvirus (KSHV) is associated with multiple myeloma (MM) remains controversial. We assayed for KSHV DNA sequences in long-term bone marrow stromal cells (BMSCs) from 26 patients with MM and 4 normal donors. Polymerase chain reaction (PCR) using primers which amplify a KSHV gene sequence to yield a 233-bp fragment (KS330233 within open reading frame 26) was negative in all cases. Aliquots of these PCR products were used as templates in subsequent nested PCR, with primers that amplify a 186-bp product internal to KS330233. BMSCs from 24 of 26 (92%) patients with MM and 1 of 4 normal donors were KSHV PCR+. DNA sequence analyses showed interpatient specific mutations (2 to 3 bp). Both Southern blot and sequence analyses confirmed the specificity of PCR results. The presence of the KSHV gene sequences was further confirmed by amplifying T 1.1 (open reading frame [ORF] K7) and viral cyclin D (ORF 72), two other domains within the KSHV genome. Immunohistochemical studies of KSHV PCR+ MM BMSCs demonstrate expression of dendritic cell (DC) lineage markers (CD68, CD83, and fascin). Serological studies for the presence of KSHV lytic or latent antibodies were performed using sera from 53 MM patients, 12 normal donors, and 5 human immunodeficiency virus (HIV)/KSHV+ patients. No lytic or latent antibodies were present in sera from either MM patients or normal donors. Taken together, these findings show that KSHV DNA sequences are detectable in BMSCs from the majority of MM patients, but that serologic responses to KSHV are not present. Ongoing studies are defining whether the lack of antibody response is caused by the absence of ongoing infection, the presence of a novel viral strain associated with MM, or underlying immunodeficiency in these patients.

Description: Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells in the bone marrow and their egress into peripheral blood with progression to plasma cell leukemia. Our previous study defined a functional role of CD40 activation in MM cell homing and migration. In this study, we examine signaling events mediating CD40-induced MM cell migration. We show that cross-linking CD40, using either soluble CD40L (sCD40L) or anti-CD40 monoclonal antibody (mAb), induces phosphatidylinositol 3–kinase (PI3K) activity and activates its downstream effector AKT in MM.1S cells. CD40 activation also activates the MAP kinase (MEK) pathway, evidenced by phosphorylation of extracellular signal-regulated mitogen-activated protein kinase (ERK), but not c-jun amino-terminal kinase (JNK) or p38, in a dose- and time-dependent manner. Using pharmacologic inhibitors of PI3K and MEK, as well as adenoviruses expressing dominant-negative and constitutively expressed AKT, we demonstrate that PI3K and AKT activities are required for CD40-induced MM cell migration. In contrast, inhibition of ERK/MEK phosphorylation only partially (10%-15%) prevents migration, suggesting only a minor role in regulation of CD40-mediated MM migration. We further demonstrate that CD40 induces nuclear factor (NF)–κB activation as a downstream target of PI3K/AKT signaling, and that inhibition of NF-κB signaling using specific inhibitors PS1145 and SN50 completely abrogates CD40-induced MM migration. Finally, we demonstrate that urokinase plasminogen activator (uPA), an NF-κB target gene, is induced by CD40; and conversely, that uPA induction via CD40 is blocked by PI3K and NF-κB inhibitors. Our data therefore indicate that CD40-induced MM cell migration is primarily mediated via activation of PI3K/AKT/NF-κB signaling, and further suggest that novel therapies targeting this pathway may inhibit MM cell migration associated with progressive MM.