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: Introduction Multiple myeloma (MM) remains incurable despite novel therapies, highlighting the need for further identification of factors mediating disease progression and resistance. Our studies have identified an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. The functional significance of increased numbers of pDCs in MM BM is evident from our observations that pDCs: are relatively resistant to novel and conventional therapies; protect tumor cells from therapy-induced cytotoxicity; promote tumor growth and survival; and suppress immune responses (Chauhan et al, Cancer Cell 2009, 16:309-323). Aberrant pDC function is evidenced in their interactions not only with MM cells, but also with other immune effector T cells and NK cells in the MM BM milieu (Ray et al, Leukemia 2015, 29:1441-1444). Directly targeting pDC interactions with MM and immune effector cells in the MM BM milieu will be required to enhance both anti-tumor immunity and cytotoxicity. However, therapies targeting pDCs are lacking. We found that IL-3R is highly expressed on pDCs, and that pDC-MM interactions trigger secretion of IL-3, which in turn, promotes both pDC survival and osteolytic bone disease. Recent efforts have led to the development of a novel therapeutic agent SL-401, which specifically targets IL-3R-expressing pDCs. Here we examined the effect of SL-401 on pDC-induced MM cell growth both in vitro and in vivo, as well as on IL-3R-expressing osteoclasts. Methods Patient MM cells, pDCs, and MNCs were obtained from normal donors or MM patients. Cell growth/viability was analyzed using MTT/WST assays. OCL function and bone resorption were measured using the OsteoAssays and TRAP staining. The RPMI-8226 cell line was used to isolate MM-SPs by flow-cytometry based Hoechst 33342 staining. SL-401 is a recombinant protein expressed in E. coli. The hybrid gene is comprised of human IL-3 fused to truncated diphtheria toxin (DT). The IL-3 domain of SL-401, which replaces the native binding domain of DT, targets SL-401 to cells that overexpress IL-3R. SL-401 was obtained from Stemline Therapeutics, NY; bortezomib, lenalidomide, pomalidomide, and melphalan were purchased from Selleck Chemicals. For animal model studies, SL-401 (16.5 μg/kg) was administered intravenously daily for 2 weeks. Results SL-401 triggered significant apoptosis in pDCs (〉95%) at low picomolar concentrations that are well within clinically achievable doses.Higher concentrations of SL-401 trigger a modest apoptosis (30%± 1.3% apoptosis at 83 ng/ml or 1.3 nM) in MM cells due to lower IL-3R expression versus pDCs. Moreover, SL-401 did not significantly induce apoptosis of normal PBMCs (8% ± 0.5% apoptosis at 83 ng/ml), suggesting a favorable therapeutic index for SL-401. SL-401 inhibited pDC-induced growth of MM cell lines and patient MM cells in a dose-dependent manner. Moreover, 6 of 9 MM samples were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Combinations of SL-401 with melphalan, bortezomib, lenalidomide, or pomalidomide induced synergistic anti-MM activity (Combination index 〈 1). SL-401 blocked monocyte-derived osteoclast formation in a dose-dependent fashion, as well as restored MM patient BM-derived osteoblast formation. Having defined the efficacy of SL-401 in targeting pDCs and pDC-triggered MM cell growth in vitro, we validated these findings in vivo using our murine xenograft model of human MM, under auspices of protocols approved by our institutional animal protection committee. SL-401 inhibited pDC-induced MM cell growth in vivo and prolonged survival in a murine xenograft model of human MM. We also evaluated the efficacy of SL-401 in vivo using our SCID-human (SCID-hu) mouse model, which reflects the human BM milieu with human cytokines and extracellular matrix proteins. SL-401 significantly abrogated pDC-triggered MM cell growth in vivo in SCID-hu model. Conclusions Our data provide the basis for using SL-401 to directly target pDCs and inhibit the pDC-MM interaction as well as target osteolytic bone disease in novel therapeutic strategies in order to enhance MM cytotoxicity, overcome drug resistance, and improve patient outcome. The interactions of immune effector cells in the MM tumor microenvironment also provide a rationale for combining SL-401 with checkpoint inhibitors. Correspondence: Dharminder Chauhan Disclosures Macri: Stemline Therapeutics, Inc., New York, NY USA: Employment. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership. Richardson:Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Chauhan:Stemline Therapeutics: Consultancy.