ALBERT

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: Abstract 2913 CRM1 (chromosomal region maintenance 1, XPO1) is the major export protein regulating degradation of key tumor suppressors by transporting them from nucleus to cytoplasm, thus abrogating their function. Highly expressed CRM1 is associated with poor prognosis in several solid tumors, and inhibition of CRM1 restores function of tumor suppressors such as p53, p21 and FOXO and IκB (cellular antagonist of NF-κB). Furthermore, CRM1 knockdown enhances sensitivity of human multiple myeloma (MM) cell lines to topoisomerase II inhibitor (Cancer Res 2009 :69, 17). In this study, we investigated a novel selective CRM1 inhibitor, KPT-185, in human MM cells. Gene expression profiling analysis using GEO database showed that CRM1 expression is significantly increased in CD138+ cells from MM patients versus monoclonal gammopathy of undetermined significance (MGUS) patients or normal donors (p

Description: Expression of B cell maturation antigen (BCMA) and secretion of its ligand a proliferation-inducing ligand (APRIL) are highly elevated in patient multiple myeloma (MM) cells and serum samples, respectively, suggesting that both proteins represent promising targets for novel immunotherapies for MM. Here, we characterize downstream molecular events following APRIL/BCMA activation in MM cells, and define functional significance of this signaling cascade in MM pathophysiology in addition to myeloma growth and survival. MM cell lines (H929, MM1S, and RPMI8226 with high, medium, and low BCMA, respectively) were first lentivirally transfected with BCMA shRNA and cDNA to knockdown and overexpress BCMA, respectively. BCMA downregulation in all MM cells significantly blocks viability and induces caspase3/7 activities, potently reducing colony formation. Conversely, BCMA overexpression in RPMI8226 (R-BCMA) cells significantly increases growth and colony formation via upregulated phosphorylation of AKT, ERK, and p65, as well as NFkB (p65, p50, p52) DNA binding activity, associated with increased expression of multiple proliferative and anti-apoptotic genes. Importantly, R-BCMA cells induce earlier onset of tumors when xenografted in mice when compared with parental cells, leading to accelerated tumor growth with increased VEGF and microvessel density (CD31+). BCMA overexpression further induces osteoclast activation factors (MIP-1α/β, SDF-1), angiogenesis factors (VEGF, CD31, IL-8), adhesion proteins (CD44, ICAM1), as well as immunosuppressive factors including PD-L1, IL-10, TGFβ. Thirty-nine IL-10 pathway associated genes are consistently elevated by BCMA overexpression in MM cells; conversely, BCMA knockdown blocks expression of these identified genes. In parallel, osteoclasts and macrophages are key sources for APRIL in the MM BM microenvironment. APRIL-triggered phosphorylation of AKT and ERK1/2 signaling, as well as NFκB DNA binding activities, are blocked by a blocking anti-APRIL monoclonal antibody hAPRIL01A. Besides the induction of similar molecular targets defined by BCMA overexpression, APRIL also significantly induces adhesion and migration of MM cells, associated with enhanced angiogenesis and adhesion/chemoattractant factors. APRIL further induces PD-L1, IL-10, and TGFβ which are blocked by hAPRIL01A. Importantly, hAPRIL01A suppresses in vivo MM cell growth within implanted human bone chips in SCID mice. It also blocks promotion of MM cell viability in cocultures with osteoclasts, macrophages, and plasmacytoid dendritic cells. Finally, lenalidomide enhances cytotoxicity of hAPRIL01A in MM cells cocultured with osteoclasts. These results define a central role of APRIL/BCMA activation in MM pathophysiology. Moreover, our study delineates their roles mediating immunosuppression in the MM BM milieu via robust upregulation of PD-L1, IL-10, and TGFβ from MM cells, strongly supporting targeting APRIL and BCMA, alone or together, in novel immunotherapies for MM. Disclosures Eenennaam: BioNovion: Employment. van Elsas:BioNovion: Employment. Munshi:Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Norvatis: Speakers Bureau.

Description: The anti-CD38 monoclonal antibody (mAb) SAR650984 (SAR) is showing promising clinical activities in ongoing trials in relapsed/refractory multiple myeloma (MM), a setting in which p53 is often mutated. Besides effector-mediated antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity, we here define additional molecular mechanisms of SAR-directed MM cell death, and further delineate enhanced anti-MM activity triggered by combining SAR with pomalidomide (Pom) or lenalidomide (Len). Since CD38 expression on MM cell lines is not as high as on primary patient MM cells, we first used lentivirus to overexpress CD38 in 3 MM cell lines with lower CD38 levels and p53 mutations. Without Fc-cross-linking agents or effector cells, SAR, but not other anti-CD38 mAb, induced homotypic aggregation (HA)-associated MM cell death in these cell lines, which was dependent on CD38 level, membrane lipid raft, and actin cytoskeleton. SAR and its F(ab)'2 fragments triggered comparable caspase 3/7 activation in CD38-highly expressing MM cells, indicating that SAR-induced caspase-dependent apoptosis is both CD38-specific and Fc-independent. Most importantly, SAR and its F(ab)'2 fragments induce lysosome-dependent cell death (LCD) in CD38 overexpressing MM cells even with p53 mutations, evidenced by enlarged lysosomes and increased lysosomal membrane permeabilization (LMP) along with leakage of cathepsin B and lysosome associated membrane protein 1 (LAMP1), in the presence or absence of IL-6 or bone marrow stromal cells. SAR significantly induced mobilization of cathepsin B and LAMP1 from lysosomes to the cytosol, particularly at the intercellular junctions, further confirming that lysosomes relocalize to sites of cell adhesion following SAR treatment. Conversely, the lysosomal vacuolar H+-ATPase inhibitor blocks SAR-induced LCD. SAR stimulated reactive oxygen species (ROS), whereas the antioxidant N-acetylcysteine (NAC) blocks SAR-induced ROS and MM cell death. Since NAC does not inhibit HA or lysosome enlargement or LMP, SAR-induced ROS occurs downstream of HA and activation of lysosomal function. SAR does not induce autophagy, indicating that autophagy is not required for this unique mode of cell death. Finally Pom, more potently than Len, enhances SAR-induced direct and indirect killing of MM cells, even resistant to Pom/Len. Moreover, combined treatment of MM cells with SAR and Pom augments caspase 3/7 activity induced by either agent alone. Taken together, these data show that SAR is the first naked therapeutic mAb with convincing direct killing of MM cell lines and patient MM cells, including those with p53 mutations and drug resistance, mainly via lysosome activation and ROS. Coupled with augmented both caspase 3/7-dependent and effector-mediated MM cytotoxicity when combined with Pom, our data further provide the framework for combination clinical trials. Disclosures Song: Sanofi: Employment. Yang:Sanofi: Employment. Adrian:Sanofi: Employment. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: 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.

Description: Abstract 1847 Aminopeptidases (AP) are necessary for the growth and development of malignant cells and have a selectively important role in the maintenance of intracellular amino acid (AA) levels in neoplastic cells. CHR2797 is a novel, low nanomolar inhibitor of the M1 family of AP, a group of metalloenzymes containing a central Zn2+ ion. CHR2797 has antiproliferative and apoptotic effects against MM in vitro by inducing the AA deprivation response (AADR). TST, an oral, chronically administered agent with a good safety profile has demonstrated activity in patients with relapsed/refractory AML and is currently under study as part of combination therapy for untreated elderly patients with AML. At the epigenetic regulatory level, Zn-dependent histone deacetylase (HDAC) cause the deacetylation of histone and non-histone cellular proteins which are critical for gene expression, inducing apoptosis and cell cycle arrest in cancer cells. LBH589 (Panobinostat) is an established pan-HDAC inhibitor with potent in vitro anti-cancer activity in many hematological malignancies. The clinical efficacy of Panobinostat is currently being studied in several Phase II/III clinical trials with particular promise seen in the treatment of MM. Here we examined the potential therapeutic effect of CHR2797, alone and with LBH589, against MM cells. Using MTS and CTG assays, CHR2797, at clinically achievable concentrations, decreased survival and proliferation in MM1S and IL-6-dependent ANBL6 cells, in the presence or absence of bone marrow stromal cells following 72 hours incubation. CHR2797 induces apoptosis in MM cells via activation of Caspase 3/7 and 9 but not Caspase 8. Significantly, CHR2797 (10 μM) induced apoptosis in patient MM cells, as seen by % of annexin V and PI from 22 + 1.5% to 39 + 2.3% after 48h incubation. Combined treatment with CHR2797 and LBH589 in MM cells (MM1S, ANBL6, and INA6) further reduced cell viability following 72 hour incubation when compared with CHR2797 treatment alone, as determined by CTG viability luminescent assay. Both drugs together also augmented growth inhibitory effects when compared with single agent alone, after 72 hours incubation followed by MTS assay. Importantly, the combination of both drugs increased caspase 3/7- & 9-mediated apoptosis than CHR2797 alone in these MM cells following 24h-treatment. Cell cycle analysis (CHR2797 at 1μM; LBH589 at 1 nM) showed an increased growth arrest in G0/G1 cells in MM1R cells treated with both drugs versus CHR2797 alone after 24 hours: 68.5±3.3% versus 36±2.5%. Furthermore, CHR2797 inhibited anti-apoptotic protein Mcl-1 in MM1R and U266 MM cells by immunoblottings. Combined treatment with CHR2797 and LBH589 further blocked Mcl-1 when compared with either treatment alone after 24 hours incubation. Together, these results show that the combination of CHR2797 and LBH589 enhanced anti-myeloma effects when compared with either drug alone. This combination, which also has the potential of being without overlapping clinical toxicities, provides a promising novel approach to anti-myeloma therapy. Disclosures: Singer: Cell Therapeutics, Inc: Employment, Equity Ownership. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees.

Description: A proliferation-inducing ligand (APRIL), a close member of B-cell-activating factor (BAFF) belonging to the TNF superfamily, is mainly produced by bone marrow (BM) accessory cells to stimulates growth and survival of multiple myeloma (MM) cells. Unlike BAFF, APRIL is dispensable in B cell homeostasis but more critical in plasma cell differentiation and survival. It has higher affinity than BAFF (nanomolar vs micromolar range) to B cell maturation antigen (BCMA) (nanomolar vs micromolar range) which expresses at high levels in all patient MM cells. APRIL also binds to a common plasma cell (PC) marker syndecan-1 (CD138) to induce signaling cascade via TACI, the other APRIL receptor in PC. We here characterize molecular mechanisms regulating APRIL activation in the BM microenvironment and further determine whether a novel anti-APRIL monoclonal antibody hAPRIL.01A inhibits its functional sequelae in MM. First, in vitro osteoclast and macrophage cultures were performed by stimulating CD14+ monocytes from MM patient samples with M-CSF/RANKL and M-CSF, respectively. Osteoclasts and macrophages secret significantly higher levels of APRIL than unstimulated CD14+ monocytes and BM stromal cells (BMSC), as confirmed by ELISA and qRT-PCR. All MM cell lines express cell surface BCMA in significantly higher level than TACI (p 〈 9.06e-15). H929 MM cells (expressing only BCMA, but not TACI), and other MM cell lines were next stimulated with APRIL, in the presence or absence of hAPRIL.01 Ab followed by immunoblotting and TaqMan® Array assays on harvested protein lysates and mRNA. APRIL stimulation consistently activated NF-kB, PI3K/AKT, and ERK1/2 signaling in MM cells. Importantly, NF-kB-DNA binding activities of p65 and p50 (p52, to a less extent), were significantly upregulated as early as 15 minute and sustain to 4h in all MM cell lines after stimulation. Conversely, hAPRIL.01 Ab completely blocked these signaling cascades, consistent with significantly decreased NF-kB-DNA binding activities in BCMA-knock-downed MM cells by shBCMA lentivirus transfection. APRIL further induced pro-survival proteins (Mcl1, Bcl2, BIRC3, XIAP) and MM cell growth-stimulating regulators (CCDN2, CDK4, CDK6, c-myc), which were completely inhibited by hAPRIL.01 Ab. These results correlated with blockade of hAPRIL.01 Ab in APRIL-promoted viability and colony formation of MM cells, in the presence of osteoclasts or macrophages. APRIL also induces adhesion of MM cells to BMSC, which was blocked by hAPRIL.01 Ab. This concurred with hAPRIL.01 Ab-reduced adhesion molecules (CD44, ICAM-1) induced by APRIL. APRIL-increased VEGF-A and PECAM-1 in MM cells was also significantly reduced by this mAb. APRIL-upregulated chemotactic/osteoclast-activating factors (MIP-1α, MIP1β, SDF-1) were also inhibited by this Ab. Other angiogenesis and adhesion/chemoattractant factors, i.e., IL-8, CXCL10, RANTES and MDC (ccl22), were changed in a similar fashion, indicating specific blocking of hAPRIL.01 Ab to APRIL-induced downstream target genes. This mAb further inhibited APRIL-increased viability of plasmacytoid dendritic cells (pDC) and diminished MM cell viability protected by pDC in 3-d cocultures. Finally, hAPRIL.01 specifically overcame APRIL-, but not IL-6, induced protection in lenalidomide- or dexamethasone-treated MM1S and H929 MM cells. These studies confirm a constitutive APRIL activation via BCMA and TACI in promoting malignancies of myeloma cells, supporting a novel therapeutics of hAPRIL.01 Ab to target MM in the BM microenvironment. Disclosures van Eenennaam: BioNovion: Employment. Elsas:BioNovion: Employment. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

Description: SAR650984 (SAR) is a naked humanized IgG1 monoclonal antibody (mAb) selectively targeting the membrane protein CD38 in early clinical development to treat multiple myeloma (MM) and other CD38+ hematological malignancies. SAR has demonstrated encouraging single agent activity in relapsed/refractory (R/R) MM patients (ASCO abstract #8532) and even better efficacy when combined with Dexamethasone and Lenalidomide (Len), without reaching a maximum tolerated dose in patients with heavily pretreated MM (ASCO Abstract #8512). It functions through multiple mechanisms including antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and direct killing against CD38-positive tumor cells including MM. Although SAR induces lysis of all CD38-expressing MM cell lines via ADCC, it only significantly induces direct killing of MOLP8 cells that express the highest CD38 surface density (~580,000/cell) among 〉 17 MM cell lines. We first sought to determine whether direct cell death induced by SAR depends on CD38 levels on MM cell membrane by generating RPMI8226 cells overexpressing CD38 (R-CD38) (Abstract #67338). R-CD38 cells express 〉 6-fold higher CD38 mRNA and surface protein levels than parental RPMI8226 cells (577,304/cell vs. 128,713/cell). Direct MM cell killing by SAR was determined using caspase 3/7 activity and CellTiter-Glo luminescent cell viability assays without goat anti-human IgG crosslinking, in the presence or absence of IL-6 or bone marrow stromal cells (BMSCs). Following overnight incubation, SAR significantly induced homotypic aggregation (HA) of R-CD38, but not control RPMI8226 cells, associated with dose-dependent activation of pro-apoptotic caspase 3/7 in R-CD38, but not control cells. Importantly, SAR decreased the viability of R-CD38, but not control cells, regardless of the presence of IL-6 or BMSCs. Direct cell death induced by SAR depends on SAR-induced HA in MM cells since SAR only blocked survival of R-CD38 and MOLP8 MM cells that show significant HA. Thus, direct apoptosis induced by SAR depends on the level of CD38 surface expression, which may contribute to clinical responses in R/R MM expressing higher CD38 levels. Next, we evaluated the combination effect of Len or Pomalidomide (Pom) with SAR on MM cells. BM mononuclear cells from MM patients were incubated with SAR (10 mg/ml) with or without 10 mM of Len or Pom overnight, followed by flow cytometric analysis to determine % Annexin V/PI staining of CD138+/BCMA+ MM cells. As expected, Pom alone induced slightly higher % of Annexin V+/PI+ MM cells than Len (41 + 1.8 % vs 49 + 1.5 %). Either combination further increased the % of double positive MM patient cells when compared with individual agent alone (from 40 + 2.1% to 70 + 3.1% combined with Len; from 40 + 2.1% to 86 + 3.4% combined with Pom). In addition, PBMC effectors from normal donors (n=4) were pretreated with Len or Pom (5 mM) for 3-7 days and used for SAR-mediated ADCC assays against MM cells (MM1S, MM1R, RPMI8226, R-38, MOLP8), with or without HS-5 or BMSCs from patients. Pom, more potently than Len, further increased SAR-induced MM cell lysis regardless of the presence of BMSCs. Moreover, additional pretreatment of MM cells with Pom overnight further enhanced SAR-induced ADCC by Pom-pretreated PBMC effectors. Both MOLP8 and R-CD38 are relative resistant to direct cytotoxicities induced by Len or Pom. Significantly, Pom, also more potently than Len, augmented direct toxicities induced by SAR in MOLP8 and R-CD38 MM cells. Taken together, we here demonstrate that SAR directly induces apoptosis of MM cells with higher CD38 levels; and that Pom, more effectively than Len, increases SAR-induced MM cell killing via apoptosis and ADCC. These data strongly support SAR as a monotherapy or in combination treatment to improve the outcome of MM patients. Disclosures Cai: Sanofi: Employment. Song:Sanofi: Employment. Yang:Sanofi: Employment. Adrian:Sanofi: Employment. Munshi:Celgene: Consultancy; Onyx: Consultancy; Janssen: Consultancy; Sanofi-Aventis: Consultancy; Ocopep: Consultancy, Equity Ownership, Patents & Royalties. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

Description: We here study targeting CD38 to overcome immunosuppression by CD4+CD25highFoxp3+ T regulatory cells (Tregs) in multiple myeloma (MM). CD38 is differentially expressed on T cell subsets with higher levels on Tregs than CD4+CD25- conventional T cells (Tcons) from MM patients vs. normal donors. CD38 levels and the percentages of CD38high Tregs are further increased by low doses of Pomalidomide (Pom) or Lenalidomide (Len), which could confer further sensitivity to CD38 targeting. This result further support combined targeting CD38 with immunomodulatory drugs (IMiDs) to mitigate tumor-related immunosuppression. Importantly, anti-CD38 mAb SAR650984 (SAR) preferentially decreases Treg while increases Tcon frequencies, which is enhanced by Pom/Len. SAR induces apoptosis and inhibits proliferation of Tregs in Fc-independent manner. It further reduces Foxp3 and IL10 in Tregs, blocks migration of Tregs, and restores proliferation and function of Tcons. Importantly, SAR augments MM cell lysis by CD8+ T and natural killer cells, as seen by enhanced cell surface CD107a for degranulation and IFNγ production. Pom/Len further enhances these effector functions induced by SAR. Ex vivo cocultures of MM cells with peripheral blood mononuclear cells (PBMCs) or Tcons significantly induce Tregs (iTregs) which express even higher CD38 than natural occurring Tregs (nTregs) in a time-dependent manner. CD38 is increased at even higher extent on iTregs induced from Tcons than PBMCs when cocultured with MM cells, indicating the conversion of Tcons into iTregs. This is associated with elevated circulating CD38+ Tregs in MM patients vs. normal donors. Besides upregulated CD38, iTregs, when compared with Tcons alone, express higher levels of CD25, Foxp3, CD44, ICOS, and PD1, while low CD127. PDL1 is concurrently increased on MM cell membrane in these cocultures. Since anti-TGFb, -PD1, or -PDL1 mAb, when added alone, partially blocks iTreg induction from Tcon, cell-cell contact via PD1/PDL1 interaction and TGFb are attributed to induction of iTregs. SAR decreases MM cell- and bone marrow stromal cell-induced iTregs and production of inhibitory cytokines TGFb and IL10, further indicating that SAR targets immunosuppressive function in CD38high iTregs. Finally, CD38 levels correlate with differential inhibition by SAR on Tregs from MM vs normal donors. Taken together, these results show that targeting CD38 can preferentially block potent immunosuppressive Tregs while restore effector function to further against MM. Disclosures Anderson: Oncoprep: Equity Ownership; Acetylon: Equity Ownership; C4 Therapeutics: Equity Ownership; Oncoprep: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Gilead: 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; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Description: Key Points FK866 combined with bortezomib induces synergistic anti-MM cell death. Addition of low doses of NAD+-depleting agent FK866 overcomes bortezomib resistance in MM cells.

Description: Key Points SIRT6 is highly expressed in multiple myeloma cells and blocks expression of ERK-regulated genes. Targeting SIRT6 enzymatic activity sensitizes multiple myeloma cells to DNA-damaging agents.