Human Multiple Myeloma Cell Lines RPMI8226 Research Articles

CD38 Is a Key Regulator of Tumor Growth By Modulating the Metabolic Signature of Malignant Plasma Cells

Introduction:Multiple myeloma (MM) is a disease of malignant plasma cells, characterized by high CD38 expression. Although the CD38-targeting monoclonal antibodies are highly effective, resistance invariably arises. Tumor CD38 levels decrease after anti-CD38 therapy, but the expression is rarely permanently silenced. This suggests that CD38 expression may offer a tumor cell survival advantage, but the direct impact of CD38 loss on tumor dynamics has not been extensively characterized.Methods:CD38 knockout (KO) cell lines were generated by CRISPR-Cas9. Immunocompetent Balb/c and immunodeficient NSG mice were injected subcutaneously with either non-targeting (NT) or CD38 KO J558 cells. Stromal adhesion was compared using labeled NT and KO cells, with OP-9 murine stroma cells. Cellular NAD content was quantified using the Promega Glo Assay. Mitochondria were isolated with the Mitochondria Isolation Kit (Thermo Scientific). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were quantified using the Seahorse Assay. Response to hypoxia was evaluated using a modular hypoxic chamber. Cell cycle was quantified using propidium iodine staining.Results:To examine the role of CD38 in murine models, we utilized the CD38-expressing, murine plasmacytoma cell line J558. Strikingly, CD38 KO cells injected into Balb/c mice demonstrated significantly decreased tumor volume compared to NT (113 mm 3 (KO) vs. 1293 mm 3 (NT) at day 25, p <0.001). In contrast, in vitro cell proliferation and colony formation between KO and NT J558 cells were nearly identical, suggesting that the effects of CD38-loss were highly context dependent. Since tumoral CD38 expression may negatively modulate the immune response, we next compared CD38 KO and NT cells injected into immunodeficient NSG mice. CD38 KOs demonstrated an approximately 2.2-fold decreased tumor volume compared to the NT (708 mm 3 (KO) vs. 1592 mm 3 (NT), p=0.07). Further examination of the role of CD38 on the immune microenvironment are ongoing. Considering that some tumor growth impairment was maintained in immunodeficient mice, we next interrogated the effect of CD38 loss on other aspects of cell proliferation using J558 as well as human MM cell lines RPMI-8226 and NCI-H929. Daratumumab induced CD38 internalization has been shown to reduce stromal adhesion of MM cells. Similarly, CD38 KO cells demonstrated reduced stromal adhesion (2.5-fold decrease for J558, p<0.005 and 2-fold decrease for H929, p<0.005). Although stroma is a known promoter of cell survival and proliferation, we further questioned whether the NAD-metabolizing activity of CD38 modulates tumor growth. CD38 overexpression can drive down intracellular NAD and impair mitochondrial biogenesis. Accordingly, we found significantly higher NAD levels in the KO J558 tumor cells compared to NT (2-fold change, p <0.05). Additionally, CD38 KO cells demonstrated significantly higher levels of mitochondrial protein compared with the NTs (5-fold in J558 and 2-fold in H929). CD38 KO cell lines also showed markedly increased metabolic activity, with nearly 2-fold increase in basal OCR and ECAR, as well as in spare respiratory and glycolytic capacity. Given the contrast between in vivo and in vitro growth capacity, we questioned whether changes in mitochondrial content and metabolic function could confer an advantage for CD38-expressing cells under conditions of hypoxia, which is an important characteristic of the tumor microenvironment. Strikingly, under hypoxia, but not normoxia, CD38 KO MM cells demonstrated significantly more cell cycle arrest, defined by G0/G1 blockage (p=0.003 for H929 and p=0.004 for RPMI).Conclusion:We have shown that CD38 KO cells demonstrate decreased tumor growth in vivo but not in vitro. While the immune modulatory potential of CD38 is recognized, some of the growth impairment we observed may be explained by non-immune mediated mechanisms such as reduced stroma adherence as well as changes in cell metabolism. Loss of CD38 was associated with increased mitochondrial respiration, but also elevated ECAR and glycolytic rate. Higher reliance on mitochondrial respiration could explain impaired CD38 KO proliferation rates under hypoxia, possibly as a result of increased generation of reactive oxygen species. DisclosuresGhiaur: Menarini Richerche: Research Funding; Syros Pharmaceuticals: Consultancy.

HIF-2α Upregulates CCR1 to Promote Dissemination of Plasma Cells in Multiple Myeloma

Introduction: Multiple myeloma (MM) disease progression and relapse is dependent on the ability of the MM plasma cells (PC) to leave the bone marrow, re-enter the circulation and disseminate to other sites throughout the skeleton. Recent data suggests that increased bone marrow hypoxia is associated with increased recirculation of MM PC (Azab et. al. Blood 2012), suggesting that hypoxia may act as a stimulus for dissemination of MM PC. However, the mechanisms responsible for this phenomenon remain unclear. In this study, we identified a potential driver of hypoxia-mediated dissemination of MM PC.Methods and results: To identify which genes were regulated by hypoxia in MM PC, we utilised microarrays to assess the effects of HIF-2α overexpression on gene expression in the human MM cell line, LP-1. The most highly upregulated gene was the C-C chemokine receptor 1 (CCR1) (6.5-fold upregulated), the receptor for C-C chemokine ligand 3 (CCL3; also known as macrophage inflammatory protein-1α [MIP-1α]), a potent inducer of MM PC migration in vitro . In addition, overexpression of HIF-2α in the human MM cell line RPMI-8226 resulted in an almost three-fold increase in migration towards CCL3, indicating that upregulation of CCR1 in response to HIF-2α confers an increase in the migratory response to CCL3.Previous studies have indicated that over two-thirds of newly diagnosed MM patients have circulating MM PC, with increased PC numbers associated with a poorer prognosis (Nowakowski et. al. Blood 2005). CCR1 expression on peripheral blood MM PC, as assessed by flow cytometry, positively correlated with the number of circulating MM PC. Moreover, in silico analysis of CCR1 expression in publically-available microarray data from newly diagnosed MM patients found that above median CCR1 expression was associated with poor prognosis in these patients in an independent dataset (p=0.025; HR=2.3 [95% CI: 1.1-4.9]; n=142 patients; E-TABM-1138).To identify the potential role of CCR1 in driving MM PC dissemination, the migration and proliferation of MM PC expressing CCR1 was assessed in vitro and in vivo . The role of CCR1 in tumour growth and dissemination of MM PC was investigated using the C57BL/KalwRijHsd mouse model of MM. In this model, luciferase-expressing mouse 5TGM1 MM PC cells were injected into the tibia, where they establish in the bone marrow and initiate systemic MM disease. Using flow cytometry, our studies show that C57BL/KalwRijHsd mice bearing 5TGM1 CCR1 overexpressing cells (5TGM1-CCR1) have increased numbers of circulating tumour cells, when normalized to tumour burden, compared with mice bearing control 5TGM1 cells. These data suggest that expression of CCR1 drives the egress of MM PC from the bone marrow into the circulation in vivo .Conclusion: This study identified CCR1 as a novel driver for hypoxia-driven MM PC migration both in vitro and in vivo . Furthermore, this study highlights an association between increased CCR1 expression, poorer prognosis and an increase in circulating tumour cells in newly diagnosed MM patients. Importantly, these findings suggest that therapies targeting CCR1 may inhibit MM PC egress from the bone marrow and limit disease progression and relapse. DisclosuresNo relevant conflicts of interest to declare.

Design, Synthesis, Molecular Docking and in vitro Evaluation of N-(4-Ethoxyphenylsulfonyl)pyrrolidine-2-carboxylic Acid Analogues as Antiangiogenic and Anticancer Agents on Multiple Myeloma

In present work, N-(4-ethoxyphenylsulfonyl)pyrrolidine-2-carboxylic acid analogs were designed, synthesized and biologically evaluated as an antiangiogenic and anticancer agent on multiple myeloma. Compounds 3i, 3k and 3m exhibited the cytotoxic action on human multiple myeloma cell line RPMI8226 with IC50 (μM) value 3.72, 3.89 2.28, respectively. These compounds possessed the antiangiogenic property and are selectively cytotoxic to cancer cells, as observed from the in vitro study of human umbilical vein endothelial cell (HUVEC) and African green monkey epithelial cell (VERO), respectively. Antiproliferative assay of the compounds on HUVECs was carried out using the dye exclusion method with trypan blue. Molecular docking study of compound 3m with vascular endothelial growth factor receptor-2 (VEGFR-2) showed possible interaction with a binding energy -62.27 kcal/mol.

Both Methylation and Copy Number Variation Participated in the Varied Expression of PRAME in Multiple Myeloma.

PurposeThe cancer-testis antigen, which is a preferentially expressed antigen of melanoma (PRAME), is an ideal target for immunotherapy and cancer vaccines. Since the expression of this antigen is relevant to therapy responses, the heterogeneity in its expression and the underlying mechanism need to be investigated.Patients and MethodsPlasma cell sorting was performed in 48 newly diagnosed multiple myeloma (MM) patients. Real-time quantitative PCR was performed to examine the PRAME transcript levels and gene copy numbers. Bisulfate clone sequencing of the PRAME promoter and exon 1b regions was performed in 4 patients. Quantitative methylation-specific PCR of the +287 CpG site was performed for all patients. The human MM cell lines RPMI8226, LP-1 and MOLP-2 were treated with 5-azacytidine.ResultsThe median PRAME transcript level was 3.1% (range: 0–298.3%) in the plasma cells sorted from the 48 MM patients. Eleven (22.9%) and 37 (77.1%) patients were individually categorized into the PRAME low- and high-expression groups according to the cut-off value of 0.05%. The methylation ratios of the promoter and the 3ʹ region of exon 1b region were both negatively related to the transcript levels. The degrees of methylation at the +287 CpG site were significantly negatively related to the transcript levels in all 48 patients (r=−0.44, P=0.0018), and those in the high-expression group (r=−0.69, P<0.0001) but not those in the low-expression group (r=−0.27, P=0.43). All 5 patients with homozygous deletions were categorized into the low-expression group. There were no significant differences in the PRAME transcript levels between the hemizygous deletion (n=8) and no deletion (n=35) groups (P=0.40). Furthermore, the PRAME transcript levels significantly increased in the MM cell lines after treatment with 5-azacytidine.ConclusionBoth methylation and copy number variation may participate in the regulation of PRAME expression in MM; in patients with no homozygous deletion, PRAME expression is mainly controlled by methylation, and a proportion of fairly low expression is caused by homozygous deletion.

Design, synthesis, and bioactivity evaluation of novel Bcl-2/HDAC dual-target inhibitors for the treatment of multiple myeloma

Multiple myeloma (MM) is the second most common haematological malignancy. Almost all patients with MM eventually relapse, and most recommended treatment protocols for the patients with relapsed refractory MM comprise a combination of drugs with different mechanisms of action. Therefore novel drugs are in urgent need in clinic. Bcl-2 inhibitors and HDAC inhibitors were proved their anti-MM effect in clinic or under clinical trials, and they were further discovered to have synergistic interactions. In this study, a series of Bcl-2/HDAC dual-target inhibitors were designed and synthesized. Among them, compounds 7e–7g showed good inhibitory activities against HDAC6 and high binding affinities to Bcl-2 protein simultaneously. They also displayed good growth inhibitory activities against human MM cell line RPMI-8226, which proved their potential value for the treatment of multiple myeloma.

Experimental study of allicin inhibiting the proliferation and invasion of human multiple myeloma cell

Objective To investigate the effect of allicin on the proliferation, apoptosis and invasion of human multiple myeloma (MM) cell line RPMI8226 in vitro, and to explore its mechanism. Methods The human MM cell line RPMI8226 cells were treated with different concentrations of allicin as 0, 5, 25, 125 μmol/L, wherein the control group was 0 μmol/L allicin treatment group. The proliferation of cells was calculated by cell counting kit-8 (CCK-8). The apoptosis and invasion ability of tumor cells were determined with flow cytometry and Transwell method respectively. The expressions of transforming growth factor-α (TGF-α), matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitor of metalloproteinase-2 (TIMP-2) protein were detected by Western blotting. Results The proliferation rates of RPMI8226 cells in groups treated with 0, 5, 25 and 125 μmol/L allicin were 1.00%±0.02%, 0.98%±0.04%, 0.73%±0.22% and 0.44%±0.15% respectively, with a significant difference (F=329.2, P<0.001). The proliferation rates of RPMI8226 cells in the 25 and 125 μmol/L allicin treatment groups were significantly inhibited compared with control group (P<0.001; P<0.001); but there was no significant difference between 5 μmol/L treatment group and control group (P=0.395). The apoptosis rates of RPMI8226 cells treated with 0, 5, 25 and 125 μmol/L allicin were 3.05%±0.53%, 4.06%±0.29%, 12.17%±1.08% and 12.81%±1.78% respectively, with a significant difference (F=531.0, P<0.001). The apoptotic rates of RPMI8226 cells in the 25 and 125 μmol/L allicin treatment groups were significantly increased compared with control group (P=0.013; P=0.012); and there was no significant difference between 5 μmol/L treatment group and control group (P=0.211). The numbers of invasive cells in the 0, 5, 25 and 125 μmol/L allicin treatment groups were 112.5±1.9, 104.8±4.0, 76.9±2.6 and 52.5±3.7 respectively, with a significant difference (F=734.9, P<0.001). The abilities of cells invasiveness in 25 and 125 μmol/L allicin treatment groups were significantly decreased compared with control group (P<0.001; P<0.001), but 5 μmol/L allicin treatment group had no significant difference compared with the control group (P=0.160). Western blotting results showed that the expression levels of TGF-α, MMP-2, MMP-9 and TIMP-2 proteins were significantly different between groups treated with different concentrations of allicin (F=227.1, P<0.001; F=348.5, P<0.001; F=359.7, P<0.001; F=158.0, P<0.001). The protein expression levels of TGF-α, MMP-2 and MMP-9 were significantly decreased in the 25 and 125 μmol/L treatment groups compared with the control group (all P<0.001), and the expression of TIMP-2 protein was significantly increased compared with the control group (all P<0.001), but there were no significant diffe-rences between the 5 μmol/L treatment group and the control group (P=0.349; P=0.744; P=0.613; P=0.567). Conclusion Allicin can significantly inhibit cell proliferation, invasive ability and induce apoptosis of human MM cell line RPMI8226 in vitro, and the mechanism may be related to inhibiting the expressions of TGF-α, MMP-2, MMP-9 and promoting the expression of TIMP-2 at the same time. Key words: Multiple myeloma; Cell proliferation; Neoplasm invasiveness; Allicin

Regulation of Osteoblast Function in Myeloma Bone Disease By Semaphorin 4D

Multiple myeloma (MM) bone disease (MMBD) is characterized by activation of osteoclasts and suppression of osteoblastic differentiation, with these changes in the bone microenvironment supporting MM cell growth and drug resistance. These complex interactions between MM cells and bone cells are incompletely understood. Current bone targeted therapy with bisphosphonates or Denosumab only blocks bone resorption but has no effect on osteoblast activity and only modest effects on MM growth. Therefore, new MMBD treatments are needed. Semaphorin-4D (Sema4D; CD100), is made by osteoclasts and inhibits osteoblasts by binding to the Plexin B receptor. Breast cancers also express Sema4d, and silencing sema4D in MDA-MB-231 breast cancer cells suppresses bone metastasis (Yang Y et al, PLoS One 2016). Since breast cancers and MM both cause osteolytic bone destruction and soluble Sema4D and Plexin B levels are increased in sera of MM patients (Terpos et al, 2012), we tested if sema4D contributed to MMBD. qPCR analysis of human MM cell lines and primary CD138+ cells showed MM cells express high levels of sema4D mRNA, comparing to the MDA-MB-231 breast cancer cells. Analysis of previously reported gene expression array data confirmed that MM cells express sema4D at a higher level compared to bone marrow plasma cells of MGUS and healthy donors (GenomicScape.com; Zhan F et al, Blood 2007; Mattiolo M et al, Oncogene, 2005). These results plus those of Terpos et al suggest that MM cells commonly express Sema4D.We next asked if the bone microenvironment increases MM expression of Sema4D. We co-cultured human MM cell lines RPMI8226 and JJN3 with mouse bones. Species -specific changes in tumor and bone were evaluated by quantitative RT-PCR. MM cells engrafted onto mouse bones, increasing markers of osteolysis similar to those seen in MM bone disease.After a week of co-culture, Sema4D expression was increased in MM cells (mean ±SD; 4.2±0.4; p=0.023), compared to MM cells grown alone. In addition, bones co-cultured with MM cells expressed higher Sema4D mRNA than bones alone (mean ±SD; 3.6±0.21; p=0.03). While co-culture increased both MM and bone Sema4D, markers of osteoblast activity, Col1a1, alkaline phosphatase and osteocalcin were suppressed. Preliminary experiments suggest that osteocytes are a major source of Sema4D expression in bone, in addition to active osteoclasts, which are much rarer cells than osteocytes. The induction of Sema4D in bone was only partially inhibited by 100nM zoledronic acid to inhibit osteoclast activity. Since osteocytes can physically interact with MM cells in vivo (Delgado Calle, Cancer Res 2016), we then tested the effect of MM cells on osteocyte sema4D expression in co-cultures of RPMI 8226 and JJN3 MM cells with MOL-Y4 osteocytic cells, separated by transwells. Both MM cell lines increased the Sema4D mRNA content of MLO-Y4 cells (mean ±SD; 3.1±0.4; p=0.036), suggesting that myeloma-secreted factors regulate osteocyte Sema4D expression.Since Sema4D is a potent osteoblast inhibitor, our data suggest that osteocyte -derived Sema4D may be a major contributor to MMBD, and that neutralization of Sema4D activity should improve the suppressed bone formation in MM. DisclosuresRoodman:Amgen: Consultancy.

Effects of INCB052793, a Selective JAK1 Inhibitor, in Combination with Anti-Myeloma Agents on Human Multiple Myeloma (MM) In Vitro and In Vivo

Introduction: Several studies have demonstrated constitutive activation of the JAK-STAT pathway in MM through dysregulated signaling of cytokines such as IL-6. In addition to its crucial role in promoting the growth, proliferation and survival of myeloma cells, IL-6 is also a potent stimulator of osteoclastogenesis and influences the tumor microenvironment in the bone marrow (BM) of MM patients by promoting an immunosuppressive milieu. Since JAK1 has been shown to be important for IL-6 signaling in MM, studies to assess the effect of JAK1 inhibition alone and in combination with other anti-MM agents were undertaken.Methods: The human MM cell lines RPMI8226 and U266 were obtained from ATCC and MM1S was kindly supplied by Steven Rosen, MD (Northwestern University, Chicago, IL). BM aspirates were obtained from patients with MM as approved by the Institutional Review Board (Western IRB BIO 001) and informed consent was obtained in accordance with the Declaration of Helsinki. BM mononuclear cells (MCs) were isolated using density-gradient centrifugation with Histopaque-1077 (Sigma-Aldrich, St. Louis, MO). All cells were maintained in RPMI1640 (Omega Scientific, Tarzana, CA) supplemented with 10% fetal bovine serum (FBS), 2mM l-glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin, in an atmosphere of 5% carbon dioxide at 37°C. Primary MM BMMCs were cultured in the presence of the JAK1 selective inhibitor INCB052793 plus a panel of anti-MM agents including the alkylating agents cyclophosphamide (CY), melphalan (MEL), and bendamustine (BEN), the proteasome inhibitor carfilzomib (CAR), dexamethasone (DEX) or the immunomodulatory agents lenalidomide (LEN) and pomalidomide (POM). Cells from RPMI8226 or U266 MM cell lines were cultured in the presence of INCB052793 plus CY, MEL, BEN, CAR, DEX, LEN, or POM. After 48 hours, cell viability was assessed using the MTS assay. For the in vivo studies, mice were implanted with a piece of the human MM tumor LAGk-1A. Seven days post-implantation, mice were randomized into treatment groups, and tumor size was measured on a weekly basis. All in vivo studies were approved by the institutional animal care and use committee.Results: In vitro studies demonstrated that combinations of INCB052793 with a broad spectrum of anti-MM agents synergistically inhibited the viability of BMMCs from MM patients. INCB052793 plus the three alkylating agents or CAR synergistically inhibited the viability of these cells. INCB052793 plus CY or MEL also significantly decreased the viability of the MM1 cell line. In vivo, LAGk-1A-bearing mice had significantly smaller tumors when treated with INCB052793 alone when compared to vehicle control at day 35 post implantation. This was in contrast to mice treated with single agent DEX, LEN or POM. Although the combination of INCB052793 with DEX, LEN or POM did not synergistically inhibit MM cell line growth in vitro, mice receiving the doublets of INCB052793 and DEX, LEN or POM demonstrated an effect on tumor growth that was superior to the doublets of DEX with LEN or POM. Mice receiving the triple combination of INCB052793 + DEX with LEN or POM demonstrated the most significant reduction in tumor growth compared with all other combinations tested. The inhibition of tumor growth with these combinations was observed throughout the study (through day 70) and all combinations were well tolerated. Concomitant with effects on tumor growth, a significant reduction in serum human IgG levels was also observed. In a separate study also using the LAGk-1A model, we evaluated the combination of INCB052793 with CAR or bortezomib (BOR). Combinations of INCB052793 + CAR or BOR were superior at inhibiting tumor growth when compared to single agent INCB052793.Conclusion: These in vitro and in vivo preclinical studies demonstrate that the combination of the JAK1 inhibitor INCB052793 with a broad spectrum of anti-MM agents are effective, and provide further support for the clinical evaluation of these drug combinations for treating MM patients. Studies to further understand the mechanistic effects of these combinations on MM signaling and the tumor microenvironment are ongoing. DisclosuresBerenson:Amgen Inc: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Immunotherapy with Long-Lived Anti-CD38 × Anti-CD3 Bispecific Antibodies Stimulates Potent T Cell-Mediated Killing of Human Myeloma Cell Lines and CD38+ Cells in Monkeys: A Potential Therapy for Multiple Myeloma

CD38, being highly expressed on malignant plasma cells, is an attractive target of new therapies for multiple myeloma (MM). Several anti-CD38 antibodies including daratumumab are in clinical development; however, a limitation of such monospecific antibodies is their inability to stimulate cytotoxic T cell killing of myeloma cells. To exploit the potent mechanism of T cell immunotherapy yet preserve the favorable drug and dosing properties of therapeutic antibodies, we designed bispecific antibodies that recruit T cells to CD38+ MM cells. Such bispecifics act via a "redirected T cell-cytotoxicity" (RTCC) mechanism because they stimulate targeted T cell-mediated killing regardless of T cell receptor antigen specificity. Unlike other bispecific formats, these antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain was also engineered to abolish binding to Fcγ receptors (to reduce the potential for nonselective T cell activation), yet preserve binding to human FcRn (to maintain long serum half-life).We first generated a library of humanized and affinity-optimized anti-CD38 × anti-CD3 antibodies and measured their potency using RTCC assays in which antibodies stimulated killing of the human MM cell line RPMI8226 by human T cells. From this screen, we selected two candidates for further assessment. XmAb13243 and XmAb13551 have 21 and 0.2 nM affinities, respectively, for human CD38, and have identical T cell-engaging domains with 8 nM affinity for human CD3. XmAb13243 stimulated RTCC with an EC50 of 2.5 ng/ml (20 pM) after 24 hr, while XmAb13551 had an EC50 of ~100 pg/ml (~1 pM).In contrast to bispecific formats lacking an Fc domain, XmAb13243 and XmAb13551 had long half-lives in mice of ~7.6 and 8.3 days, respectively. Because these bispecifics were optimized for human CD38 and CD3 binding and do not crossreact with mouse antigens, we next evaluated efficacy in immunodeficient SCID mice engrafted with human PBMCs. In this model, engrafted human B cells differentiate into CD38+ plasma cells, which produce high levels of human Ig. Bispecific antibodies dosed at 0.2, 1, and 5 mg/kg, 7 and 15 days after engraftment, suppressed human IgG2, IgM, and IgE to below detectable levels by Day 14 (> 50-fold for IgG2, > 1,000-fold for IgM, and > 80-fold for IgE). Daratumumab at 5 mg/kg was markedly less potent than bispecifics, reducing IgG2 by 2-fold, IgM by 6-fold, and IgE by 3-fold. The control bispecific anti-RSV × anti-CD3 (which binds to T cells but not to CD38+ cells) had no effect on IgG2, IgM, or IgE levels. To investigate activity against an immune response requiring production of new human plasma cells, mice were vaccinated with tetanus toxoid 8 days after engraftment. Anti-CD38 × anti-CD3 bispecifics suppressed human anti-tetanus antibody titers to baseline (> 100-fold), while daratumumab suppressed titers by only 2-fold.We next assessed efficacy in cynomolgus monkeys. Unlike daratumumab, which does not crossreact with monkey CD38, XmAb13243 and XmAb13551 bind to both CD38 and CD3 in monkeys (23 and 0.3 nM, respectively, to CD38, and 6 nM to CD3 for both). We treated monkeys with a single dose of XmAb13243 or XmAb13551 at 2, 5, and 20 μg/kg. T cells were activated within 1 hr, as measured by dramatic increases in CD25 and CD69 activation markers. Within 8 hr, T cells depleted circulating CD38+ cells by > 95% at the 20 μg/kg dose.Our results demonstrate that XmAb13243 and XmAb13551 effectively recruit T cells to kill CD38+ cells in vivo. Our preclinical data in monkeys and humanized mice provide a rationale for clinical testing of anti-CD38 × anti-CD3 bispecific antibodies in patients with multiple myeloma and other CD38+ malignancies. DisclosuresChu:Xencor: Employment, Equity Ownership. Miranda:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

Very Late Antigen-4 (α4β1 Integrin) Targeted PET Imaging of Multiple Myeloma

Biomedical imaging techniques such as skeletal survey and 18F-fluorodeoxyglucose (FDG)/Positron Emission Tomography (PET) are frequently used to diagnose and stage multiple myeloma (MM) patients. However, skeletal survey has limited sensitivity as it can detect osteolytic lesions only after 30–50% cortical bone destruction, and FDG is a marker of cell metabolism that has limited sensitivity for intramedullary lesions in MM. Targeted, and non-invasive novel probes are needed to sensitively and selectively image the unique molecular signatures and cellular processes associated with MM. Very late antigen-4 (VLA-4; also called α4β1 integrin) is over-expressed on MM cells, and is one of the key mediators of myeloma cell adhesion to the bone marrow (BM) that promotes MM cell trafficking and drug resistance. Here we describe a proof-of-principle, novel molecular imaging strategy for MM tumors using a VLA-4 targeted PET radiopharmaceutical, 64Cu-CB-TE1A1P-LLP2A. Cell uptake studies in a VLA-4-positive murine MM cell line, 5TGM1, demonstrated receptor specific uptake (P<0.0001, block vs. non-block). Tissue biodistribution at 2 h of 64Cu-CB-TE1A1P-LLP2A in 5TGM1 tumor bearing syngeneic KaLwRij mice demonstrated high radiotracer uptake in the tumor (12±4.5%ID/g), and in the VLA-4 rich organs, spleen (8.8±1.0%ID/g) and marrow (11.6±2.0%ID/g). Small animal PET/CT imaging with 64Cu-CB-TE1A1P-LLP2A demonstrated high uptake in the 5TGM1 tumors (SUV 6.6±1.1). There was a 3-fold reduction in the in vivo tumor uptake in the presence of blocking agent (2.3±0.4). Additionally, 64Cu-CB-TE1A1P-LLP2A demonstrated high binding to the human MM cell line RPMI-8226 that was significantly reduced in the presence of the cold targeting agent. These results provide pre-clinical evidence that VLA-4-targeted imaging using 64Cu-CB-TE1A1P-LLP2A is a novel approach to imaging MM tumors.

Preclinical Efficacy of the Investigational Orally Bioavailable Proteasome Inhibitor MLN9708 in Myeloma Bone Disease

AbstractAbstract 4014 Introduction:Bone destruction, a hallmark of multiple myeloma (MM), arises as a consequence of the interactions between MM cells and the bone marrow microenvironment, which lead to an increase in the bone-resorptive activity and number of osteoclasts (OC) and a reduction of the bone-forming activity and differentiation of osteoblasts (OB).MLN9708, which hydrolyzes to pharmacologically active MLN2238 in aqueous solution, is an investigational proteasome inhibitor (PI) with demonstrated preclinical anti-myeloma activity. However, it is currently not known whether MLN9708, may have a beneficial effect on myeloma-associated bone disease. Here, we have conducted in vitro and in vivo studies to evaluate its ability to promote osteogenic differentiation and to inhibit OC formation and function in the myeloma setting. Patient samples, material and methods:The human MM cell lines RPMI-8226 and MM.1S (or RPMI-8226-luc and MM.1S-luc) together with the mesenchymal stem hMSC-TERT cell line were employed. Also, MSCs from BM samples of healthy donors and MM patients were used in OB differentiation studies, whereas PBMCs from healthy volunteers were used to generate OCs. NOD.SCID.IL2Rγ−/− mice were used in the in vivo model of disseminated human MM. MLN2238 and bortezomib (Velcade) were provided by Millennium Pharmaceuticals, Inc.OB differentiation from MSCs and OB function were investigated by measurement of ALP activity, quantitative mineralization, luciferase reporter assays, siRNA gene silencing and real time RT-PCR. The effect of the new PI on OC formation was assessed by enumeration of multinucleated (≥3) TRAP-positive cells. Measurement of resorbed area, immunofluorescence and flow cytometry were used to further investigate the effect of MLN2238 on OC function. In our in vivo model, bioluminescence imaging, micro-CT analysis and serum levels of Igλ and bone markers were determined. Results:Physiologic concentrations of MLN2238 were able to stimulate the osteogenic differentiation of MSCs from both myeloma patients and healthy donors in vitro to an extent comparable to bortezomib; this was assessed by increased levels of ALP activity, higher expression of bone formation markers (Runx2, osterix, osteopontin and osteocalcin) and augmented matrix mineralization. The enhanced OB formation and function induced by MLN2238 was at least partly due to induction of T-cell factor 4 (TCF4) transcriptional activity, as well as to activation of the unfolded protein response. A similar range of MLN2238 doses also markedly inhibited OC formation and resorption from human progenitors. Similarly to that described with bortezomib, MLN2238 treatment of human pre-OCs prevented RANKL-induced NF-κB activation, disrupted the integrity of the F-actin ring and also reduced the expression of the αVβ3 integrin, thus contributing to inhibition of OC function. MLN2238 was also able to overcome the growth advantage conferred to MM.1S-luc cells by co-culture with MSCs or OCs.Oral administration of MLN2238 in a mouse model of disseminated human MM decreased human RPMI-8226-luc tumor burden as assessed by diminished bioluminescence signal and decreased serum levels of Igλ secreted by RPMI-8226-luc cells. In addition, MLN2238 prevented tumor-associated bone loss with significant increases in femoral trabecular bone parameters as compared to vehicle control animals. Serum markers of bone turnover showed that MLN2238 inhibited bone resorption (decreased levels of CTX) while enhancing bone formation (increased levels of P1NP). Conclusion:MLN2238 in vitro was capable of promoting osteoblastogenesis and OB activity as well as of inhibiting OC formation and function to an extent similar to bortezomib. In a disseminated human MM mouse model, orally administered MLN2238 showed anti-resorptive and bone-anabolic effects in addition to its anti-tumor properties. Given the thus far available data on the preclinical safety and favorable pharmacologic properties of MLN2238, it is conceivable that MLN9708, the clinical formulation of this proteasome inhibitor, may also achieve bone benefits in myeloma patients. Disclosures:Berger:Millennium Pharmaceuticals, Inc.: Employment. San-Miguel:Millennium Pharmaceuticals, Inc.: Consultancy.

The HDAC Inhibitor LBH589 Enhances the Antimyeloma Effects of the IGF-1RTK Inhibitor Picropodophyllin

We have previously shown the use of the insulin-like growth factor type 1 receptor tyrosine kinase (IGF-1RTK) inhibitor picropodophyllin (PPP) as an attractive strategy to combat multiple myeloma (MM) in vitro and in vivo. After a combinatorial drug screening, the histone deacetylase inhibitor LBH589 was shown to act in synergy with PPP reducing survival of MM cells. In this study, we tried to elucidate the molecular mechanisms underlying this combinatorial effect. The in vitro anti-MM effects of PPP and LBH589 alone and in combination were evaluated by studying apoptosis, cell cycle distribution, and downstream transcriptome using both human MM cell lines and cells from the murine 5T3MM model. In vivo the effect on survival of 5T33MM-inoculated mice was evaluated. In the human MM cell line RPMI8226, treatment with PPP and LBH589 in combination resulted in a five-fold increase of apoptosis, and an additive effect on the cleavage of the active forms of caspase-8 was observed as compared with the single drug treatments. Cell cycle analysis revealed an accumulation of cells in the G(2)-M phase and subsequent downregulation of cell cycle regulating proteins. These data were also confirmed in the 5T33MM cells in vitro. Also, the transcriptome was analyzed by Affymetrix arrays showing gene expression alterations mainly in categories of genes regulating apoptosis and cell adhesion. Combined treatment in vivo resulted in a significantly prolonged survival of 5T33MM-inoculated mice. The results indicate an improved MM treatment opportunity in using a combination of PPP and LBH589.

The proteasome inhibitor bortezomib disrupts tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and natural killer (NK) cell killing of TRAIL receptor-positive multiple myeloma cells

Bortezomib, a potent 26S proteasome inhibitor, is approved for the treatment of multiple myeloma (MM) and clinical trials are under way to evaluate its efficacy in other malignant diseases. However, cytotoxic effects of bortezomib on immune-competent cells have also been observed. In this study, we show that bortezomib downregulates cell surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on primary human interleukin (IL)-2-activated natural killer (NK) cells. Pharmacological inhibition of the transcription factor, NF-κB also profoundly decreased TRAIL expression, suggesting that NF-κB is involved in the regulation of TRAIL expression in activated human NK cells. Furthermore, perforin-independent killing of the human MM cell lines RPMI8226 and U266 by NK cells was markedly suppressed following bortezomib treatment. In addition, blocking cell surface-bound TRAIL with a TRAIL antibody impaired NK cell-mediated lysis of the TRAIL-sensitive MM cell line, RPMI8226. In conclusion, the proteasome is likely to be involved in the regulation of TRAIL expression in primary human IL-2-activated NK cells. Proteasome inhibition by bortezomib disrupts TRAIL expression and TRAIL dependent and/or independent pathway-mediated killing of myeloma cells, suggesting that bortezomib may potentially hamper NK-dependent immunosurveillance against tumors in patients treated with this drug.

In vitro study of human multiple myeloma cell transfected with soluble vascular endothelial growth factor receptor-1 gene

Vascular endothelial growth factor (VEGF) is the most important angiogenic factor of multiple myeloma (MM). This study was to investigate the effect of transfection of human soluble vascular endothelial growth factor receptor-1 (sFlt-1) gene on the proliferation of human MM cell line RPMI8226. The recombinant plasmid pcDNA3-sFlt-1 was constructed and transfected into RPMI8226 cells. The expression of sFlt-1 was identified by reverse transcription-polymerase chain reaction (RT-PCR) and ELISA. The effects of sFlt-1 protein on the proliferation and VEGF expression of RPMI8226 cells were investigated by MTT assay and ELISA, respectively. The recombinant plasmid pcDNA3-sFlt-1 was successfully transfected into RPMI8226 cells. sFlt-1 protein expression was identified by ELISA, which inhibited the proliferation of RPMI8226 cells and reduced VEGF concentration in the culture supernatant. RPMI8226 cells can express sFlt-1 protein with high biological activity when transfected with the sFlt-1 gene, which inhibits the proliferation of RPMI8226 cells.