Multiple Myeloma Cell Lines RPMI8226 Research Articles

Cancerous Inhibitor of PP2A Silencing Inhibits Proliferation and Promotes Apoptosis in Human Multiple Myeloma Cells.

Multiple myeloma is the second most prevalent type of blood cancer, representing approximately 1% of all cancers and 2% of all cancer deaths. There is therefore a strong need to identify critical targets in multiple myeloma neoplasia and progression. Cancerous inhibitor of PP2A (CIP2A) is a human oncoprotein that regulates cancer cell viability and anchorage-independent growth and induces apoptosis. The present study investigated CIP2A function in the human multiple myeloma cell lines RPMI-8226 and NCI-H929 to determine whether it can serve as a potential therapeutic target. CIP2A was silenced in the cells by transfection of short interfering RNA and cell proliferation and apoptosis were evaluated by a tetrazolium salt-based assay and flow cytometry, respectively. CIP2A knockdown inhibited proliferation and induced apoptosis in RPMI-8226 and NCI-H929 cells and decreased the phosphorylation of phosphoinositide 3-kinase (PI3K) p85, AKT1, and mammalian target of rapamycin (mTOR) without affecting total protein levels. Treatment of CIP2A-depletion cells with insulin-like growth factor 1 decreased the effects of CIP2A inhibition on cell viability and apoptosis. These results indicate that CIP2A modulates myeloma cell proliferation and apoptosis via PI3K/AKT/mTOR signaling and suggest that it can potentially serve as a drug target for the treatment of multiple myeloma.

Tuning T Cell Affinity Improves Efficacy and Safety of Anti-CD38 × Anti-CD3 Bispecific Antibodies in Monkeys - a Potential Therapy for Multiple Myeloma

CD38 is highly expressed on plasma cells and is an attractive target for multiple myeloma (MM) therapies. Several anti-CD38 antibodies including daratumumab and SAR650984 show promising results in clinical development, though such antibodies are not able to stimulate T cell-mediated killing of myeloma cells. To exploit a T cell immunotherapy mechanism while retaining the favorable drug properties of therapeutic antibodies, we designed bispecific antibodies that recruit T cells to CD38+ MM cells. Such bispecifics act via redirected T cell-cytotoxicity (RTCC) to stimulate T cell-mediated target cell killing regardless of T cell receptor antigen specificity. These anti-CD38 × anti-CD3 antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain lacks binding to Fcγ receptors to minimize nonselective T cell activation, yet retains binding to FcRn to maintain long serum half-life.We have previously reported that XmAb13551, a humanized and affinity-optimized anti-CD38 × anti-CD3 antibody, stimulates killing of the CD38+ MM cell line RPMI8226 by human T cells and suppresses human Ig levels in SCID mice engrafted with human PBMCs, showing much greater efficacy than daratumumab in these models (Blood 2014 124:4727). We also investigated efficacy of XmAb13551 in monkeys given a single dose of 2, 5, and 20 μg/kg. Within 1 hr after dosing, CD25 and CD69 activation markers were upregulated on T cells and within 8 hr, circulating CD38+ cells were depleted by > 95% at the 20 μg/kg dose. However, depletion of peripheral CD38+ cells was not sustained, suggesting that a large antigen sink was limiting drug exposure. Although higher dosing might overcome an antigen sink, higher doses of XmAb13551 (0.2 mg/kg or higher) resulted in a T cell-mediated cytokine release syndrome (CRS) in monkeys.We reasoned that an anti-CD38 × anti-CD3 antibody with reduced CD3 affinity would stimulate sufficient RTCC to deplete MM cells, yet would attenuate the acute T cell activation (and associated CRS) induced by high-affinity coengagement of T cells with CD38+ target cells. Using XmAb13551 as a starting point, we engineered a series of bispecifics retaining the same high-affinity (0.2 nM) binding to CD38, but with reduced affinity to CD3. We selected two antibodies - XmAb15426 and XmAb14702 - that have significantly reduced CD3 affinity. As expected, these molecules showed reduced potency in RTCC assays using T cells to kill RPMI8226 cells, with potency correlating with CD3 affinity (XmAb13551 > XmAb15426 >> XmAb14702).We next tested XmAb15426 and XmAb14702 at single doses of 0.5 mg/kg and 3 mg/kg, respectively, in cynomolgus monkeys. Both antibodies were well-tolerated at these higher doses, consistent with the moderate levels of IL6 observed in serum from the treated monkeys. Moreover, XmAb15426, with intermediate CD3 affinity, more effectively depletes CD38+ cells at 0.5 mg/kg compared to the original high-affinity XmAb13551 dosed at 2, 5 or 20 µg/kg. Depletion by XmAb15426 was more sustained compared to the highest dose of XmAb13551 in the previous study (7 vs. 2 days, respectively). Notably, although target cell depletion was greater for XmAb15426, T cell activation (CD69, CD25 and PD1 induction) was much lower in monkeys treated with XmAb15426 even dosed 25-fold higher than the 20 µg/kg XmAb13551 group. XmAb14702, with very low CD3 affinity, had little effect on CD38+ cells and T cell activation.Our results demonstrate that modulating T cell activation by attenuating CD3 affinity is a promising method to improve the therapeutic window of T cell-engaging bispecific antibodies. This strategy has potential to expand the set of antigens amenable to targeted T cell immunotherapy by improving tolerability and enabling higher dosing to overcome antigen sink clearance with targets such as CD38. We have shown that by reducing affinity for CD3, XmAb15426 effectively depletes CD38+ cells while minimizing the CRS effects seen with comparable doses of its high-affinity counterpart XmAb13551. Our preclinical data for XmAb15426 provide a rationale for clinical testing of this bispecific antibody in patients with multiple myeloma and other CD38+ malignancies. DisclosuresMoore:Xencor, Inc.: Employment, Equity Ownership. Lee:Xencor, Inc.: Employment, Equity Ownership. Schubbert:Xencor, Inc.: Employment, Equity Ownership. Miranda:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Ardila:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Chu:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

Phospho-Flow Cytometry Quantitation of STAT Signalling Checkpoints in Malignant and Non-Malignant Cells Identifies Patients Suitable for pSTAT3 and 5 Targeted Inhibitors

BackgroundIL-6 receptor/JAK/STAT is a major signalling pathway associated with pleiotropic functions including cell transformation and proliferation. STAT3, one of a family of 7 STAT members, sits at the apex of the signalling cascade and is a potential target for cancer therapy. Small molecular weight inhibitors either prevent STAT3 phosphorylation or target high constitutive levels of phosphorylated pSTAT3 (pSTAT3). Increased constitutive expression of pSTAT3 is a hallmark of solid tumors and is associated with increased morbidity. However recent studies suggest that this may not apply to hematological malignancies. Phospho-flow cytometry offers a novel approach for the quantitation of constitutive and cytokine induced pSTAT3 expression in subsets of hemopoietic cells. Constitutive pSTAT3 expression is not a prognostic factor for patients with either multiple myeloma (MM) or acute myeloid leukemia (AML), though we recently demonstrated that IL-6 induced pSTAT3 was associated with better prognosis in MM (χ2 =13.06; p<0.0003) (Leukemia 2015; 29:483) . There is considerable heterogeneity of pSTAT3 expression between patients which suggests that quantitation of these new biomarkers may help to identify those patients most likely to respond to STAT-targeted therapy. MethodsWe have determined constitutive and IL-6 induced pSTAT3 and 5 in malignant plasma cells of patients with MM (n=25) and for comparison, the blast cells in AML (n=8) and CD5+ B cells in chronic lymphocytic leukemia (CLL) (n=12) as well as the non-malignant B and T cells in each sample. Normal BM cells (n=9) served as a control. We have also determined pSTAT3 and 5 expression in "side population" cells (SP), the putative myeloma stem cells, and determined whether MM and AML tumor cells induce pSTAT3 on normal lymphocytes in vitro by co-culture experiments. IL-6 receptor expression was also determined on all cells.ResultsConstitutive pSTAT3 expression was increased (>5% of normal lymphocytes) in the malignant plasma cells of 44% of patients with MM but by comparison was not increased in AML or B-CLL. Constitutive pSTAT5 was increased (>5%) in 68% of MM plasma cells and 75% of AML cells and was either normal or decreased in CLL cells. Patients with increased pSTAT3 or 5 in MM cells also had high expression in autologous normal T cells. The level of constitutive pSTAT3 and 5 was not related to the disease status (relapse vs remission). In patients with MM, IL-6 induced an increased pSTAT3 expression (induced/constitutive ratio >1.5) in both MM cells and T cells in 88% of samples. IL-6 induced pSTAT3 expression in blasts of 89% of AML patients (median ratio = 79). There was no correlation between the level of pSTAT3 induced by IL-6 and IL-6 receptor expression. IL-6 did not increase pSTAT5 levels in MM, AML or CLL. SP cells in the MM cell line RPMI 8226 had lower constitutive pSTAT3 than the non-SP population and a lower IL-6 receptor expression, but had a greater response to cytokine stimulation, suggesting that SP cells may be more sensitive than non-SP cells to inhibitors of STAT phosphorylation. Addition of RPMI 8226 cells or supernatant to normal T cells caused an increase in pSTAT3, suggesting that the upregulation of pSTAT3 is at least partly due to a tumor-derived soluble agent in the microenvironment.ConclusionsThese results demonstrate the heterogeneity of constitutive and cytokine induced pSTAT3 and 5 expression in MM, AML and CLL. Elevated levels of constitutive pSTAT3 (approx 44% of MM) and pSTAT5 (88% of MM and AML) in the malignant clone were associated with high levels in autologous non-malignant T cells and are not of prognostic significance. Therefore elevated pSTAT3 and 5 may not be an exclusive hallmark of the malignant clone, as reported in solid tumors, but rather a result of the microenvironment and a high IL6-R expression. Phospho-flow cytometry enables precise quantitation of constitutive and cytokine-induced pSTAT3 and 5, and thus can identify the patients most likely to respond to direct STAT-3 or 5 inhibitor therapies. SP cells (putative MM stem cells) appear to be potentially targetable. Inhibitors targeting signalling pathways may be more efficacious if therapy is directed to patients with targetable checkpoints. DisclosuresHart:DendroCyte BioTech Pty Ltd: Equity Ownership.

E-Selectin Ligand Expression Increases with Progression of Myeloma and Induces Drug Resistance in a Murine Transplant Model, Which Is Overcome By the Glycomimetic E-Selectin Antagonist, GMI-1271

There is increasing evidence that E-selectin and its ligands play an important role in the progression of multiple myeloma (MM) and drug resistance. We reported that the sialyltransferase ST3GAL6 influences homing and survival in MM, and postulated that it may function in the synthesis of E-selectin ligands (Glavey et al Blood, 2014). We also found that a small subpopulation of cells (~ 5%) from MM cell lines express functional E-selectin ligands, which could be expanded under hypoxic conditions typical of the bone marrow (BM) microenvironment. These cells were identified by reactivity with an antibody (HECA452), which binds the same carbohydrate epitope required for binding to E-selectin. Rolling of MM1S cells on E-selectin was blocked by a small molecule glycomimetic antagonist to E-selectin (GMI-1271). Moreover, GMI-1271 significantly enhanced the anti-myeloma activity of bortezomib (BTZ) in an in vivo murine transplant model (Natoni et al Blood, 2014). We now extend these observations to obtain a more complete understanding of the role E-selectin plays in MM biology and chemotherapy resistance for its potential clinical relevance.The parental, heterogeneous MM cell lines MM1S and RPMI8226 (MM1Spar, RPMI8226par, respectively) were sequentially sorted to obtain cell lines highly enriched (>85% and 80%) for the expression of cell surface carbohydrates bound by HECA452, and designated MM1SHECA452 and RPMI8226HECA452. The cell lines could be passaged in vitro and were stable for enriched E-selectin ligand expression. In contrast to parental cells, both MM1SHECA452 and RPMI8226HECA452 showed strong binding to E-selectin in static adhesion assays. Both MM1SHECA452 and RPMI8226HECA452 exhibited strong rolling on E-selectin under shear stress. MM1Spar or RPMI8226par failed to roll well on E-selectin. The addition of GMI-1271 during culture conditions led to a marked reduction in adhesion of MM1SHECA452 and profoundly inhibited rolling on E-selectin of both HECA452 enriched MM cell lines.The significance of these in vitro findings was studied in vivo. MM1Spar or MM1SHECA452 cells were injected i.v. into SCID beige mice. Beginning 5 days post tumor injection, the survival impact of treatment with saline control, GMI-1271, BTZ or a combination of both was determined. Mice transplanted with MM1SHECA452 had more aggressive disease with significantly shorter survival compared to those transplanted with MM1Spar. In contrast to MM1Spar cells, mice engrafted with MM1SHECA452 demonstrated a marked resistance to BTZ treatment. Whereas GMI-1271 treatment alone had no impact on survival, the combination of GMI-1271 and BTZ led to a highly significant improvement in survival of MM1Spar engrafted mice (P=0.0363), and more importantly broke the resistance and restored the anti-myeloma activity of BTZ in MM1SHECA452 engrafted mice (P=0.0123) (figure 1). The number of peripheral blood (PB) human CD138+ cells was increased in MM1SHECA452-engrafted mice within 60 min following a single injection of GMI-1271, and persisted for at least 24 hours (2.37% v. 0.03%, p <0.001). This effect was consistent with GMI-1271 disrupting the tumor microenvironment and mobilizing MM1SHECA-452 cells from the BM niche.Given these findings we wished to see if samples from patients with MM express E-selectin ligands and whether higher levels are seen with disease progression. BM and/or PB were obtained following informed consent from patients with MM and plasma cells (CD38+/CD138+) were analyzed for E-selectin ligand expression by flow cytometry using the HECA452 antibody. To date all primary MM samples (n=25) contained HECA452-reactive cell populations (median 22%). A consistently higher proportion of circulating MM cells express HECA452 when compared with paired BM samples (n=14), with a median difference of 33% (Wilcoxon signed rank test, p=0.02). HECA452 expression of MM in PB was significantly higher (on average 40% higher) in samples taken at relapse vs. diagnosis, (unpaired t test, p = 0.0008)These data provide compelling evidence that E-selectin ligand bearing cells play an important role in disease progression and drug resistance in MM, and a strong rationale for clinical strategies incorporating GMI-1271 to improve patient outcome. [Display omitted] DisclosuresSmith:GlycoMimetics, Inc.: Employment. Locatelli-Hoops:GlycoMimetics, Inc.: Employment. Oliva:GlycoMimetics, Inc.: Employment. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. O'Dwyer:Celgene: Honoraria, Research Funding.

Lenalidomide affect expression level of cereblon protein in multiple myeloma cell line RPMI8226.

We investigated the mechanisms of action of immuno-modulatory drug (lenalidomide) on the protein expression of cereblon (CRBN) and their therapeutic targets in the multiple myeloma cell line RPMI8226. The multiple myeloma cell line RPMI8226 was cultured and treated with different concentrations of lenalidomide and bortezomib to determine the proliferation inhibition rate, apoptosis rate, and protein expression of CRBN. The results revealed that both lenalidomide and bortezomib inhibited the proliferation of RPMI8226 and promoted cell apoptosis. However, the protein expression of CRBN decreased signifi-cantly after treatment with lenalidomide, while bortezomib had no effect on the expression of CRBN. We confirmed that CRBN may be a target of lenalidomide.

Effects and mechanism of arsenic trioxide in combination with rmhTRAIL in multiple myeloma

The anti-tumor potential of arsenic trioxide (ATO) and recombinant mutant human TRAIL (rmhTRAIL) has been confirmed in various kinds of tumors. However, the effects and mechanism of the two drugs in combination in multiple myeloma (MM) have not been established. In this study, we evaluated the proliferation inhibition and apoptosis induction effects of ATO and rmhTRAIL as single agents and in combination on the MM cell lines RPMI8226 and U266. Then, we used high-performance liquid chromatography and mass spectrometry to find differentially expressed proteins before and after drug treatment and to analyze the mechanism underlying the effect of ATO and rmhTRAIL on MM cells. Results indicated that ATO and rmhTRAIL had synergistic or additive effects on proliferation inhibition and apoptosis induction in MM cells, and the mechanism underlying the two-drug combination might involve regulation of the expression of several proteins affected by ATO and rmhTRAIL. Our study provides evidence of a potential new combination treatment strategy for MM.

TRAF6 is required for BLyS-mediated NF-κB signaling in multiple myeloma cells.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the IL-1R/TLR and TNFR superfamilies to the transcription factors NF-κB and AP1. Elevated expression of the TNF family member B-lymphocyte stimulator (BLyS) in multiple myeloma (MM) has been described recently. However, the precise process by which BLyS signals in myeloma cell remains unknown. Here, we identified increased expression of TRAF6 in MM patient cells and the MM cell lines U266, RPMI8226, and KM3. Furthermore, rhBLyS induced TRAF6 up-regulation in these cells in a dose-dependent manner. Both the classical and alternative NF-κB pathways were activated by rhBLyS treatment. Depletion of TRAF6 by siRNA decreased levels of p-p65 and p-p100, even after stimulation with rhBLyS. Down-regulation of TRAF6 also abrogated rhBLyS-mediated cell viability. These findings suggest that TRAF6 is required for BLyS-mediated NF-κB signaling in myeloma cells and is a potential molecular therapeutic target in MM.

Analysis of the anti-proliferative and the pro-apoptotic efficacy of Syk inhibition in multiple myeloma.

BackgroundMultiple myeloma (MM) is a clonal B cell malignancy characterized by proliferation of malignant plasma cells in the bone marrow. Despite high-dose melphalan therapy with autologous stem cell transplantation (ASCT) and the introduction of immunomodulatory drugs like bortezomib or lenalidomide, that have been associated with improved survival, MM is still incurable and new treatment options are needed. In B cell malignancies such as chronic lymphocytic leukaemia (CLL) or diffuse large B cell lymphoma (DLBCL), Syk (spleen tyrosine kinase) inhibitors have shown promising in vitro and first clinical results. In our study, we analyzed the potential of Syk as a target in MM.MethodsThe MM cell lines AMO-1, U266 and RPMI8226 and primary MM cells were treated with the Syk inhibitors BAY61-3606, R406 or Piceatannol and proliferation, migration and apoptosis induction were analyzed. Effects on involved intracellular signaling cascades were determined by Western blotting. Furthermore, we analyzed synergistic and additive effects of Syk inhibitors in combination with established anti-myeloma drugs and experimental inhibitors (e.g. PI-3-Kinase inhibitor NVP-BEZ235).ResultsIncubation of MM cell lines as well as primary MM cells with Syk inhibitors resulted in a reduced proliferation and stromal cell-derived factor-1 alpha (SDF-1 alpha) induced migration that was accompanied by a concentration dependent inhibition of the MAP-Kinase, characterized by reduced phosphorylation of ERK an p38 molecules, and NF-kappaB signalling pathways. Furthermore, Syk inhibition induced apoptosis in MM cells in a dose-dependent manner, characterized by reduced expression of pro-caspase 3, increased PARP-1 cleavage and enhanced release of cytochrome c. In addition combined treatment of MM cells with Syk inhibitors and NVP-BEZ235 (dual PI3-kinase/mTOR inhibitor) or MAPK inhibitors (PD98059, SP600125, U0126, SB203580) resulted in increased apoptotic activity of the drugs.ConclusionsOur results show that Syk inhibition might represent a promising new treatment option in MM with an increased efficacy when combined with MAP kinase inhibitors. Furthermore, our study strongly underlines the potency of Syk inhibitors as a potential therapeutic treatment option for MM patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s40164-015-0016-z) contains supplementary material, which is available to authorized users.

Abstract 3815: Synergistic mechanisms of oncolytic reovirus with bortezomib in overcoming therapy resistance of multiple myeloma

Abstract Introduction: Multiple myeloma (MM) is an incurable hematologic malignancy that accounts for 10% of hematopoietic neoplasms. The potential of reovirus (RV) as a novel therapeutic agent for MM under in vitro, in vivo and ex vivo conditions has been demonstrated by us and a phase I clinical trial of MM with RV therapy has shown indications of efficacy. Recently we have shown that RV could synergize with several MM drugs such as bortezomib (Bort), cafilzomib, dexamethasone and perifosine and importantly, enhance its therapeutic potential in therapy resistant MM. Here we show that RV/Bort synergy in MM is mediated via enhanced apoptosis than augmented viral progeny production. Methods: MM cell lines RPMI 8226, U266, NCIH929, U266, INA-6, KMS11 and OPM2 were incubated with live or dead RV at a MOI of 40 for 24, 48, and 72 hours respectively and cell death was assessed via the WST assay. RV resistant OPM2 and moderately resistant KMS-11 were used in combination therapy and treated with RV and drugs at various concentrations to evaluate Effective Dose 50% (ED50) values and synergism was determined by the Chou-Talalay method. To assess viral progeny production, MM cells were treated with RV+/- drugs and harvested at various time points and frozen. Following 3 freeze thaw cycles viral progeny was assessed via plaque titration on L929 cells. To assess apoptosis, cells treated with RV or Bort singly or in combination were harvested 48 hours and stained with AnnexinV/FITC and 7AAD and analyzed by flow cytometry. Results: RV sensitive RPMI8226 cells demonstrated the maximal viral progeny production. Log-fold increases in viral plaques forming units (PFU)/ml detected at 72h was 3.04 for RV+Bort treated cells and 3.58 for RV alone treated cells indicating productive virus replication in both treatments. RV resistant OPM2 cells demonstrated the least amounts of virus progeny (0.99-1.28 PFU/ml fold increase) in RV+Bort treated or RV alone treated cells respectively. KMS11 cells treated with RV alone showed a log-fold increase in virus progeny of 2.32PFU/ml at 72h. Interestingly, when KMS11 cells were treated with RV+Bort no significant increase in RV progeny was seem at 72h indicating minimal RV replication in these cells in the presence of Bort. The synergistic cytotoxicity observed with RV+Bort in all 3 cell lines was due to enhanced apoptosis where significantly augmented apoptosis was seen in combination treatment than in single agent treatment. Conclusion: Not all MM cell lines are amenable to RV mediated cell death. This has important implications for the future use of RV as a therapeutic agent. Mechanisms such as autophagy and necroptosis that may contribute to RV/drug mediated cytotoxicity is currently been investigated and experimentation in animal models is also underway. Understanding the signalling pathways of resistant tumour will help develop a more personalized approach of RV therapy for MM patients in the future. Citation Format: Chandini M. Thirukkumaran, Zhong-Qiao Shi, Joanne Luider, Karen Kopciuk, Paola Neri, Nizar Bahlis, Don Morris. Synergistic mechanisms of oncolytic reovirus with bortezomib in overcoming therapy resistance of multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3815. doi:10.1158/1538-7445.AM2015-3815

Abstract 4282: All-trans retinoic acid enhances the effector functions of the anti-CD38 antibody SAR650984

Abstract SAR650984 is a humanized IgG1 monoclonal antibody selectively targeting CD38 that is in clinical development for the treatment of multiple myeloma (MM). SAR650984 eliminates MM cells through different mechanisms, including antibody dependent cytotoxicity and phagocytosis (ADCC and ADCP, respectively), complement dependent cytotoxicity (CDC), and direct induction of apoptosis. The aim of this study was to assess the cell killing activity of SAR650984 following genetic and chemical manipulation of CD38 expression in MM cells. We established several MM cell lines engineered to overexpress CD38; median levels of CD38 were 537,000 receptors/cell, versus 29,000 receptors/cell in the parental cell lines. ADCC, ADCP, CDC, and apoptosis induced by SAR650984 treatment were all significantly increased in the CD38-overexpressing lines compared with the parental cell lines. Based on these data, we hypothesized that pre-treatment of MM cells with all-trans retinoic acid (ATRA) would enhance the activity of SAR650984, since ATRA can induce up-regulation of CD38 expression. Treatment of the MM cell lines JJN-3, ARH-77, U266, Karpas-620, SKMM-2, RPMI-8226, NCI-H929, KMS-12PE and MOLP-8 with ATRA resulted in a dose-dependent 1.4- to 6.7-fold up-regulation of cell surface expressed CD38. Pretreatment of MM cells with ATRA for 48 hours resulted in enhanced SAR650984-mediated ADCC and ADCP compared to non-pretreated cells. The enhancement of ADCC and ADCP activity by ATRA pretreatment was recorded in all the cell lines tested. In conclusion, these in vitro data are supportive of further evaluation of the combination of SAR650984 and ATRA for the treatment of MM. Citation Format: Lucas Bush, David Harper, Rita Greco, Zhili Song, Guang Yang, Francisco Adrian. All-trans retinoic acid enhances the effector functions of the anti-CD38 antibody SAR650984. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4282. doi:10.1158/1538-7445.AM2015-4282

Effect of PI3Kδ inhibitor CAL-101 on myeloma cell lines and preliminary study of synergistic effects with other new drugs

To investigate the proliferation inhibitory role and mechanism of PI3Kδ inhibitor CAL-101 on multiple myeloma (MM) cells, and to provide new therapeutic options for MM treatment. MM cell lines U266 and RPMI8226 cells were treated with various concentrations of CAL-101. MTT assay and CalcuSyn software were performed to determine the inhibitory effect of CAL-101 and the synergistic effect with PCI- 32765, SAHA (suberoylanilide hydroxamic acid), BTZ (Bortezomib) on MM cells. The protein expression level of p-AKT, p-ERK, AKT, ERK and PI3Kδ processed by CAL-101 were analyzed by Western blot. CAL-101 at concentration of 15, 20, 25, 30 and 40 μmol/L could induce significant dose-dependent proliferation inhibition on U266 cells after treatment for 48 hours. The cell proliferation inhibition rates were (33.54 ± 1.23)%, (41.72 ± 1.78)%, (53.67 ± 2.01)%, (68.97 ± 2.11)% and (79.25 ± 1.92)%, respectively. Similar results were found in RPMI8226 cell line. Western blots showed high expression level of p-AKT, p-ERK, AKT, ERK and PI3Kδ in cell lines and MM primary cells. p-AKT and p-ERK protein expression levels were down-regulated significantly by CAL-101 treatment. Synergistic effect has been verified between CAL-101 and PCI-32765, SAHA and Bortezomib in U266 cell line, and PCI-32765, Bortezomib in RPMI8226 cell line with CI values less than 1. CAL-101 could inhibit proliferation of MM cell lines. High levels of p-AKT, p-ERK, AKT, ERK and PI3Kδ protein expression were observed in both cell lines and primary cells. Down-regulation of p-AKT and p-ERK probably related with the mechanism of CAL-101 in MM cell proliferation inhibition. CAL-101 has significant synergistic effect with PCI-32765, SAHA and BTZ.

Inhibitory effects of parthenolide on the activity of NF-κB in multiple myeloma via targeting TRAF6.

This study examined the mechanism of the inhibitory effect of parthenolide (PTL) on the activity of NF-κB in multiple myeloma (MM). Human multiple myeloma cell line RPMI 8226 cells were treated with or without different concentrations of PTL for various time periods, and then MTT assay was used to detect cell proliferation. Cell cycle and apoptosis were flow cytometrically detected. The level of protein ubiquitination was determined by using immunoprecipitation. Western blotting was employed to measure the level of total protein ubiquitination, the expression of IκB-α in cell plasma and the content of p65 in nucleus. The content of p65 in nucleus before and after PTL treatment was also examined with immunofluorescence. Exposure of RPMI 8226 cells to PTL attenuated the level of ubiquitinated Nemo, increased the expression of IκB-α and reduced the level of p65 in nucleus, finally leading to the decrease of the activity of NF-κB. PTL inhibited cell proliferation, induced apoptosis and blocked cell cycle. Furthermore, the levels of ubiquitinated tumor necrosis factor receptor-associated factor 6 (TRAF6) and total proteins were decreased after PTL treatment. By using Autodock software package, we predicted that PTL could bind to TRAF6 directly and tightly. Taken together, our findings suggest that PTL inhibits the activation of NF-κB signaling pathway via directly binding with TRAF6, thereby suppressing MM cell proliferation and inducing apoptosis.

Establishment of a human multiple myeloma xenograft model in the chicken to study tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA). In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Effect of shRNA-mediated silencing of BDNF gene on VEGF expression of RPMI8226 cells

目的研究脑源性神经营养因子（BDNF）对多发性骨髓瘤（MM）细胞表达血管内皮生长因子（VEGF）的调控作用，初步探讨BDNF与MM血管新生的关系。方法设计和构建针对人BDNF基因的siRNA表达载体，用脂质体Lipofectamine™2000介导转染，将空载体质粒pGenesil-1和重组质粒pGenesil-shRNA-BDNF分别转染MM细胞株RPMI8226细胞（分别命名为P0组、P1组）。采用RT-PCR法和Western-blot法鉴定干扰后BDNF的表达；MTT法和流式细胞术检测细胞的增殖、凋亡；RT-PCR法和ELISA法检测干扰BDNF对细胞表达VEGF的影响。结果①成功构建了针对BDNF的siRNA真核表达载体。与未转染组（Pn组）及P0组相比，P1组BDNF mRNA水平及蛋白水平显著下调（P值均<0.05）；②P1组细胞的增殖活性（0.42±0.06）显著低于Pn组（0.56±0.06）和P0组（0.50±0.04）（P值均<0.05）；③P1组细胞的早期凋亡率[（53.84±9.95）%]明显高于Pn组[（5.23±2.46）%]和P0组[（9.10±3.46）%]（P值均<0.01）；④P1组细胞VEGF121、VEGF145和VEGF165 mRNA表达水平分别为Pn组的（0.62±0.07）、（0.47±0.09）和（0.57±0.02）倍（P值均<0.05）。⑤P1组细胞VEGF的分泌水平分别为Pn组的（0.36±0.05）倍和P0组的（0.44±0.06）倍（P值均<0.05）。结论BDNF基因沉默可促进RPMI8226细胞凋亡并抑制其增殖，下调RPMI8226细胞VEGF的表达，BDNF可能通过调控MM细胞VEGF的表达而参与MM血管新生的病理过程。

Cytokine-induced apoptosis inhibitor 1 inhibits the growth and proliferation of multiple myeloma.

The present study investigated the differential expression of cytokine-induced apoptosis inhibitor 1 (CIAPIN1) in human multiple myeloma (MM) bone marrow tissue and adjacent healthy bone marrow tissue. In addition, the effect of a transduced CIAPIN1 gene on the growth of the RPMI-8226 human MM cell line was investigated. CIAPIN1 protein expression was detected in 32 samples of paraffin-embedded MM and adjacent healthy bone marrow tissue using immunohistochemistry. The CIAPIN1 gene (Ad-CIAPIN1, small interfering RNA) was inserted into a lentiviral vector and transfected into the RPMI-8226 human MM cell line. The expression of target proteins CIAPIN1 and insulin-like growth factor 1 U (IGF-1), cell cycle-regulatory proteins and functional proteins was detected using western blotting. MTT and soft agar colony formation assays were conducted, and cellular tumorigenicity in nude mice was assessed, in order to investigate the proliferative capacity of cells in vitro and in vivo. Flow cytometry was applied in order to analyze changes in the cell cycle and cell apoptosis. CIAPIN1 expression was significantly reduced in cells from the 32 MM samples compared with those from healthy bone marrow (P<0.05). Upregulation of CIAPIN1, following transduction by lentiviral vectors, caused cells to arrest in G1/S phase of the cell cycle and significantly inhibited the growth of the RPMI-8226 MM cell line in vitro and in vivo. CIAPIN1 was shown to inhibit cell growth. Specifically, it inhibited cyclin-dependent kinase 2, cyclin-dependent kinase 4 and insulin-like growth factor-1. Increased expression of CIAPIN1 also led to an increase in the levels of p27 and Rb, an effect that may have been achieved via regulation of cell cycle proteins and functional proteins. The results of the present study suggest that downregulation of the CIAPIN1 gene in MM cells may be associated with the development of this disease. CIAPIN1 transfection in RPMI-8226 cells significantly inhibited the growth of tumor cells, suggesting that the CIAPIN1 gene is a potential tumor suppressor.

Engagement of CD99 Reduces AP-1 Activity by Inducing BATF in the Human Multiple Myeloma Cell Line RPMI8226.

CD99 signaling is crucial to a diverse range of biological functions including survival and proliferation. CD99 engagement is reported to augment activator protein-1 (AP-1) activity through mitogen-activated protein (MAP) kinase pathways in a T-lymphoblastic lymphoma cell line Jurkat and in breast cancer cell lines. In this study, we report that CD99 differentially regulated AP-1 activity in the human myeloma cell line RPMI8226. CD99 was highly expressed and the CD99 engagement led to activation of the MAP kinases, but suppressed AP-1 activity by inducing the expression of basic leucine zipper transcription factor, ATF-like (BATF), a negative regulator of AP-1 in RPMI8226 cells. By contrast, engagement of CD99 enhanced AP-1 activity and did not change the BATF expression in Jurkat cells. CD99 engagement reduced the proliferation of RPMI8226 cells and expression of cyclin 1 and 3. Overall, these results suggest novel CD99 functions in RPMI8226 cells.

Activation of Rac1 through FARP1 Induces Dexamethasone Resistance in Multiple Myeloma Cells

Recently, novel agents such as bortezomib and lenalidomide have been introduced for multiple myeloma (MM) treatment and have improved patients’ survival drastically. However, dexamethasone remains a mainstay in the treatment of MM. Dexamethasone effectively induces tumor cell death when used for the initial treatment of MM. In addition, dexamethasone has a synergistic effect with novel agents and is hence used in combination with such agents. However, prolonged dexamethasone exposure may lead to drug resistance. To elucidate the mechanism of dexamethasone resistance, we generated a dexamethasone-resistant subline of the MM cell line RPMI8226. We cultured RPMI8226 cells with 1 µM dexamethasone for 7 weeks and established the dexamethasone-resistant cell line Dex-R. This cell line showed no difference in survival in the presence or absence of 1 µM dexamethasone. We then examined differences in gene expression between RPMI8226 and Dex-R cells using cDNA microarray. Expression of the FARP1 gene, which is a transforming growth factor beta (TGF-b) target gene in myeloma cells, was increased approximately 50-fold in Dex-R cells compared to that in RPMI8226 cells. In some myeloma patients who become chemoresistant, myeloma cells show high levels of FARP1 expression at the initial stage. FARP1 has a Rho-GEF domain and can associate with proteins on the cell membrane through the FERM domain. In the nervous system, FARP1 is involved in synaptogenesis via the activation of Rac1. Based on these observations, we hypothesize that Dex-R cells acquires dexamethasone resistance with an increase in the level of FARP1 expression via the activation of Rac1. To verify this hypothesis, we established inducible FARP1 knockdown Dex-R cells using the TET-ON lentiviral system. We cultivated these cells for 24 h with doxycycline and added 1 µM dexamethasone. A total of 48 h after adding dexamethasone, we measured cell viability using the MTS assay. We cultured Dex-R cells with a Rac1 inhibitor (NSC23766) and added dexamethasone 12 h later. FARP1 expression decreased to approximately 10% in FARP1 knockdown cells 24 h after the addition of doxycycline. Without dexamethasone, there was no difference in survival in the presence or absence of doxycycline. However, when cells were cultured with dexamethasone, the growth of FARP1 knockdown Dex-R cells was significantly inhibited compared with that of the control (Fig 1). Next, we examined the change in dexamethasone resistance on the addition of the Rac1 inhibitor. The number of cells increased after 96 h without dexamethasone. On the other hand, the number of cells significantly decreased when cultured with dexamethasone (Fig 2). These data suggest that resistance to dexamethasone in Dex-R cells was mitigated by the inhibition of Rac1. We conclude that the activation of Rac1 through FARP1 is one mechanism of dexamethasone resistance in MM. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Modulation of CD38 Expression Levels on Multiple Myeloma Tumor Cells By All-Trans Retinoic Acid Improves the Efficacy of the Anti-CD38 Monoclonal Antibody Daratumumab

▪INTRODUCTION Daratumumab is an anti-CD38 monoclonal antibody (mAb) with lytic activity against multiple myeloma (MM) cells, including ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). In current clinical phase I/II trials, daratumumab induced anti-MM activity; however, the depth of the response varied between patients. Up till now it is unknown what determines the intrinsic sensitivity of MM cells towards daratumumab-mediated ADCC and CDC. We examined potential determinants of daratumumab sensitivity including CD38 levels, the frequency of effector cells, and expression levels of the complement inhibitory proteins, CD46, CD55, and CD59, which interfere with the different steps of complement activation.RESULTS In bone marrow samples from MM patients, we observed a significant correlation between CD38 expression and daratumumab-mediated ADCC (127 patients; R = 0.428; P < 0.0001) as well as CDC (56 patients; R = 0.338; P = 0.011). Similarly, experiments with isogenic MM cell lines expressing different levels of CD38, revealed that the level of CD38 expression correlates with the extent of daratumumab-mediated ADCC and CDC. Other determinants of daratumumab susceptibility include the effector:target ratio for ADCC, and levels of the complement-inhibitory proteins CD55 and CD59 for CDC. Our data suggest that upregulation of CD38 expression may improve the anti-MM activity of daratumumab. Since, interaction of all-trans retinoic acid (ATRA) with nuclear retinoic acid receptors results in altered expression of target genes including induction of CD38 expression, we evaluated the combination of ATRA and daratumumab. As little as 10 nM ATRA was sufficient to induce a 1.9 – 4.4-fold increase in CD38 expression on the MM cell lines RPMI8226, UM9, and XG1, which resulted in a significant improvement of daratumumab-mediated ADCC and CDC. Importantly, 10 nM ATRA alone resulted in no or only a minimal increase in MM cell death. In addition, ATRA induced a 1.0 – 26.5 (median 1.7) fold increase in CD38 expression on primary MM cells from 26 patients. Also in these primary MM cells, pretreatment with ATRA resulted in a significant increase in their susceptibility to daratumumab-mediated CDC in 13 out of 16 patients as well as ADCC in 8 out of 11 patients. ATRA also enhanced the efficacy of daratumumab in MM cells which are completely resistant to daratumumab-mediated CDC and/or ADCC. Pooled results of these patients show that ATRA improved CDC mediated by 10 µg/mL daratumumab from 16.1 % to 43.9 % (P < 0.0001), and ADCC from 25.1 % to 39.5 % (P = 0.0315). Importantly, expression levels of CD55 and CD59 on MM cells were also significantly reduced by ATRA, which may explain that ATRA improves CDC to a higher extent than ADCC.CONCLUSION Our results provide evidence that CD38 expression levels may predict response to daratumumab. Furthermore, we show that ATRA increases CD38 expression on MM cells, resulting in enhanced daratumumab-mediated lysis of MM cells. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients. DisclosuresLokhorst:Celgene: Research Funding; J&J: Research Funding; Genmab: Research Funding. Martens:J&J: Research Funding. Doshi:Janssen R&D: Employment. Mutis:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding. Sasser:Janssen R&D: Employment. van de Donk:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding.

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.

Anti-Myeloma Activity by the Combination of the JAK2 Inhibitor Ruxolitinib with Lenalidomide and Corticosteroids

Introduction: The JAK2 inhibitor ruxolitinib (RUX) is an inhibitor of the Janus kinase family of protein tyrosine kinases (JAKs) that is effective for the treatment of myeloproliferative diseases. Immunomodulatory drugs (IMiDs) including lenalidomide (LEN) and corticosteroids have shown efficacy for the treatment of multiple myeloma (MM). The JAK-STAT signaling pathway plays key roles in the growth and survival of malignant plasma cells in MM. In this study, we evaluated the preclinical anti-MM effects of RUX in combination with LEN and corticosteroids, both in vitro and in vivo, and in a patient with MM and polycythemia rubra vera (PRV).Methods: The human MM cell lines U266, RPMI8226 and MM1S cells were derived from ATCC. Primary MM tumor cells were isolated from MM patients’ bone marrow aspirates. The cells were seeded at105 cells/100ul/well in 96-well plates and incubated for 24 h in the presence of vehicle, RUX, LEN or dexamethasone (DEX) alone, RUX + LEN, RUX + DEX, or all three drugs together for 48 h. Cell viability was quantified using the MTS cell proliferation assay. In vitro, synergy between ruxolitinib and lenalidomide or dexamethasone was assessed using the median effect method of Chou and Talalay. For the in vivo studies, the human myeloma tumors (LAGκ-1A or LAGκ-2) were surgically implanted into the left superficial gluteal muscle of anaesthetized naive SCID mice. Mice were blindly assigned to one of the experimental groups, and treatment was initiated 7–21 d after tumor implantation. LEN was administered via oral gavage daily (30 mg/kg). RUX (3 mg/kg) was given via intraperitoneal (IP) injection twice daily. Dexamethasone was administered daily (1.5mg/kg) via IP injection. An 88 year old MM patient with PRV who developed MM on RUX alone and then progressed on LEN+DEX was treated with the combination of all three drugs.Results:In vitro, RUX induced concentration-dependent inhibition of viability in all three MM cell lines (U266, RPMI8226 and MM1S) at RUX 50 mM and inhibition of primary MM tumor cells at a higher concentration (100 mM). In contrast, RUX had negligible cytotoxic effects on normal peripheral blood mononuclear cells (PBMCs). We next examined cell viability in the presence of RUX plus LEN or DEX. First, U266 cells were incubated with a fixed concentration of LEN (30 mM) or DEX (40 mM) with increasing concentrations of RUX (0.1–100 mM) for 48 h. At RUX 50 mM, the cytotoxic effects of LEN were enhanced and at RUX 1 mM, the anti-myeloma effect of DEX was increased. Moreover, the cytotoxic effects of RUX, LEN and DEX were greater than RUX in combination with either LEN or DEX in U266 cells. Similar results were obtained using the RPMI8226 and MM1S cell lines as well as primary MM tumor cells. Next, we evaluated RUX in combination with lenalidomide and dexamethasone in vivo using SCID mice bearing either the human LAGκ-1A or LAGκ-2 MM xenografts. RUX (3mg/kg), LEN (15mg/kg) or DEX (1mg/kg) alone did not inhibit tumor growth in either mice bearing LAGκ-1A or LAGκ-2. In contrast, the combination of RUX with DEX but not LEN slightly decreased tumor volume. However, the combination of all three drugs at the same doses showed a marked reduction of tumor size and delay of tumor growth in both human MM xenograft models. In addition, a patient with MM and PRV experienced sustained and ongoing reductions in his serum M-protein, IgG, and 24-urine M-protein with achievement of a partial response on low doses of RUX (2.5 mg twice daily), LEN (2.5 mg daily), and methylprednisolone (20 mg daily) that has been ongoing for more than 12 months after developing MM on RUX alone and then progressing on the combination of LEN and methylprednisolone.Conclusion: This study illustrates that the combination of the JAK2 inhibitor RUX, LEN and corticosteroids shows both preclinical and promising clinical results for the treatment of MM. DisclosuresNo relevant conflicts of interest to declare.