Multiple Myeloma Cell Lines U266 Research Articles

Rocaglamide breaks TRAIL-resistance in human multiple myeloma and acute T-cell leukemia in vivo in a mouse xenogtraft model

Multiple myeloma (MM) is an incurable malignancy by the presently known therapies. TRAIL is a promising anticancer agent that virtually not shows any toxicity to normal cells. We have recently carried out clinical trials with a human circularly permuted TRAIL, CPT, against MM saw a partial response in approximate 20–30% of patients. In the current study, we investigated the cause of CPT resistance and revealed that the majority of the MM patients express elevated levels of c-FLIP. Knockdown of c-FLIP expression by siRNA alone was sufficient to increase CPT-mediated apoptosis in a CPT-resistant human MM cell line U266. To overcome CPT resistance, we investigated the combination of CPT with Rocaglamides(s) in MM which has been shown to inhibit c-FLIP expression in vitro. We show that Rocaglamide(s) overcomes CPT resistance in U266 in vitro and significant increases in anti-tumor efficacies of CPT in mice xenografted with U266. Similar results were also obtained in mice xenografted with the CPT-resistant human acute T-cell leukemia cell line Molt-4. Our study suggests that the combination of Rocaglamide(s) with CPT may provide a more efficient treatment against myeloma and leukemia.

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.

Anti-Cancer Effects of Clofazimine As a Single Agent and in Combination with Cisplatin in Multiple Myeloma

Multiple myeloma (MM) is a malignant neoplasm of bone marrow plasma B cells with high morbidity. Clofazimine (CLF) is an FDA-approved leprostatic, anti-tuberculosis, and anti-inflammatory drug that was previously shown to have growth suppression effects on various cancer types such as hepatocellular, lung, cervix, esophageal, colon, and breast cancers as well as melanoma, neuroblastoma, and leukemia cells. The objective of this study was to evaluate the anticancer effect of CLF on U266 resistant MM cell line.The relative cell viability of a panel of hematological cell lines (Jurkat, U266, Namalwa, K562, HL60) treated with 10 µM CLF after 24 h of treatment significantly reduced the viability in all cell lines, with percentages ranging between 28% (U266) and 38% (Jurkat) (p<0.001). IC50 value of CLF was found as 9.8 ± 0.7 µM on the U266 cell line. Previous studies showed that this level of CLF does not inhibit growth of healthy cells, which supports safety of CLF. CLF had both dose (2, 5, 10 µM) and time (12, 24, and 48 h) dependent growth inhibitory effect. Combination chemotherapy is an approach to increase the effectiveness of chemotherapeutics as well as overcome drug resistance and suppresses side effects of drugs. Therefore, we evaluate the combination effect of CLF in U266 cells and showed that combination with cisplatin led to a synergistic interaction between two compounds in all tested dose regimes, resulting in a 2.5-7.1 fold marked increase in cell death. Importantly this synergism was observed in U266 cells, which have mutant p53 at A161T showing resistance to cytotoxic agents such as platinum analogs (cisplatin etc.). <>Depolarization of the mitochondrial membrane is one of the first events in apoptosis. JC-1 is a lipophilic and cationic dye that reversibly changes color from green to red as the mitochondrial membrane potential increases (depolarization). JC-1 assay used in both flow cytometry analyses and fluorescence microscopy images have shown that relative to the control, CLF treatment results in the depolarization of mitochondrial membrane 15, 20.5, 14.3 fold respectively at 12, 24, and 48 h in U266 cell line (Figure 1).The caspase family of cysteine proteases plays an important role in apoptosis. Caspase-3 is a major protease activated during the early stages of programmed cell death. 10 µM CLF was applied for 12, 24, and 48 h and anti-active caspase-3 PE stained U266 cells were analyzed by flow cytometry. Caspase-3 activity is enhanced 5.6, 24.5 and 13.6-fold relative to untreated controls at 12h, 24h and 48 h respectively.Phosphatidylserine (PS) translocation to the outer leaflet of the cellular membrane is one of the key steps in early stages of apoptosis. To support our previous findings on apoptotic effect of CLF, we employed Annexin-V assay. CLF treatment caused a significant increase in the percentage of early and late apoptotic cells at 12 h (2.1 and 1.8 fold respectively), 24 h (4.1 and 12.3 fold) and 48 h (10.1 and 11.5 fold). Fluorescence microscopy images also supported flow cytometry data (Figure 2). Collectively, all three apoptosis assay results show that CLF significantly induces apoptosis in U266 cells.Our study is the first to show apoptotic and growth inhibitory effects of CLF on a p53-mutant resistant MM cell line U266. Our results also proved that combined therapy employing CLF together with chemotherapeutics seems to be a possible future therapeutic approach for MM. Further in vivo and clinical studies are warranted to evaluate its therapeutic potential for resistant MM treatment. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interleukin-6 Drives Multiple Myeloma Progression By up-Regulating of CD147/Emmprin Expression

Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation of plasma cells in the bone marrow (BM). CD147, also known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD147 is expressed in a variety of tissues, and is involved in a number of physiological and pathological processes. With regard to MM, it was recently documented that the aberrantly elevated expression of CD147 has been tightly correlated with MM cell colonization and proliferation. However, it is still unclear what mechanism is involved in the dysregulation of CD147 expression in MM. In this study, we found that CD147 can be up-regulated by interleukin-6 (IL-6), which is derived from either autocrine or paracrine sources and plays an essential role in the malignant progression of MM. When serum-starved MM cell lines RPMI8226, MM1.S and NCIH929 were stimulated with 20ng/mL of IL-6 for 24 hours, we found that the expression of CD147 on MM cell membrane was significantly up-regulated, as determined by flow cytometric analysis. Moreover, when using the IL-6 autocrine MM cell line U266, CD147 level was found to be continuously increased in serum-starved MM cells in a time-dependent manner. Interestingly, the increase of CD147 in U266 cells was found to be abrogated when using neutralizing antibody to block the secreted IL-6. Next, we investigated the molecular mechanisms involved in the IL-6-mediated upregulation of CD147 in MM cells. We confirmed that IL-6 can activate the JAK/STAT3 signaling pathway, inducing the phosphorylation of STAT3 at Tyrosine 705 in RPMI8226 and MM1.S cells. Further studies using ChIP analysis demonstrated that there is a STAT3 binding site at the position −659 to −650 in the promoter of the CD147 gene. In addition, when inhibiting of STAT3 phosphorylation with a specific inhibitor S3I-201, the CD147 expression in MM cells was accordingly decreased. Finally, we found that siRNA mediated knock-down of CD147 in RPMI8226 and MM1.S cells can decrease the proliferation and migration of MM cells induced by IL-6 and Cyclophilin A (CypA). In conclusion, our data provide evidence that IL-6 can up-regulate CD147 expression in a STAT3-dependent manner, and offer a compelling rationale for exploring this axis as a therapeutic target for MM. DisclosuresNo relevant conflicts of interest to declare.

Myeloma-Derived Exosomes and Soluble Factors Suppress Natural Killer Cell Function

IntroductionThe complex makeup of the tumor microenvironment (ME) exerts selective pressure on cancer cells leading to immune escape, and novel immunotherapeutic interventions have emerged from evolving knowledge of the immune system and tumor cells. Natural killer (NK) cells are innate immune cells that exert potent anti-tumor effects. Previously we have reported that ex vivo expansion of NK cells by co-culture PBMCs with K562mbIL15-41BBL can generate large numbers of highly active expanded NK cells (ENKs). These ENKs expand further upon adoptive transfer in vivo both in a murine model and in patients (Garg et al. 2012, Szmania et al. 2015), and have been shown to persist and retain their cytolytic ability. We are currently applying ENK therapy in a Phase II clinical trial at our institute in gene expression profiling-defined high-risk multiple myeloma (MM), a patient population which fares poorly despite the use of novel drugs and autologous stem cell transplantation.A potential obstacle to successful NK cell-based therapies is the suppression of NK cells in the MM bone marrow ME (BM-ME) by immunosuppressive cells, various soluble factors (SF), microRNAs, and exosomes. Exosomes are endosomal-derived, 30-130nm microvesicles present in almost all body fluids. Their number is significantly higher in cancer patients. Tumor-derived exosomes contain a wide range of bioactive molecules, such as microRNA, RNA, DNA and protein, and play a major role in immune escape, promoting tumor progression. Their size, structure, and presence in serum allow them to transport their cargo to distant targets. This study was designed to characterize the potential adverse effects of myeloma-derived exosomes (MEXs) and myeloma-derived SF (MSF) on NK cell function and determine if such inhibition can be overcome by cytokine support.MethodsMEXs were isolated from OPM2 myeloma cell line-derived conditioned media (MCM) using the Total Isolation Reagent (Life Technologies, Carlsbad CA). Transmission electron microscopy (TEM), flow cytometry, and western blot (WB) analysis were used for characterization of exosomes. Fresh NK cells (non-activated) and ENKs were incubated with MCM or MEXs and evaluated for their viability and cytolytic ability in standard 4-hour chromium release assays. Flow cytometry was used to evaluate the immunophenotype of these cells, including activation, costimulatory, inhibitory receptors, and adhesion molecules.ResultsTEM confirmed the presence of exosomes in MCM (size and morphology). Interestingly, OPM2-derived MEXs did not express the exosome-specific marker CD9, but did express CD63, and CD81. Flow cytometry showed that MEXs contain MICA/B, TGFβ, TRAIL-R1, TRAIL-R2, MHC class I, HLA-E, and ICAM3. NK cells exposed to MEXs demonstrated a dose-dependent, significant decrease in specific lysis of the MM cell lines JJN3, OPM2, and U266 in cytotoxicity assays compared to control NK cells (13%-51%, p<0.0005). In addition, a time-dependent decrease in NK cell-mediated lysis was observed in these MM cell lines at 24hours (14%-34%) versus 48hours (30%-48%; p<0.0005). A similar downward trend in the activity of ENKs incubated with MEXs was also noted but to a lesser extent. We hypothesize that highly-activated ENKs are able to partially overcome MEX-mediated inhibition compare to resting NK cells. We also noted a considerable decrease in the cytolytic ability of ENKs incubated with MCM which contains suppressive soluble factors in addition to MEXs (28%-58%, p<0.0005). Further, this suppression in ENK activity was partly rescued by fresh IL2 incubation (18-36%, p<0.01). Many of the activating receptors (NKp46, NKp30, NKp44, NKG2D), costimulatory receptors (2B4, NTB-A, NKp80, DNAM-1), activation markers (CD26, CD69), and adhesion molecules (LFA-1, CD54) were down regulated on the ENK cells incubated with MCM. However, differences were not as significant in these receptors on ENK cells incubated with MEX.ConclusionMEXs and other SF released from myeloma cells are capable of modulating the function and phenotype of NK cells and ENKs. MCM is more immunosuppressive as it contains both MEX and MSF. Cytolytic ability of ENKs could be partially restored by incubation in fresh IL2 medium. Further characterization of MEXs and MCM by proteomics is in progress. (Data will be presented). DisclosuresDavies:Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Morgan:Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding; Univ of AR for Medical Sciences: Employment; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

In-Vitro and in-Vivo Synergistic Effect of Melphalan and Dual DNA Repair Inhibition in Multiple Myeloma

High dose melphalan is commonly used in patients with multiple myeloma (MM). Resistance to melphalan has been linked to the ability to repair DNA damage. To test whether DNA repair inhibitors overcome resistance to melphalan and and also have a direct anti-MM effect, we tested MM cell lines RPMI8226 and U266 in-vitro and in-vivo, using a NOD/SCID/ gamma null (NSG) xenograft model.RPMI8226 and U266 cells were initially treated in-vitro with the PARP inhibitor ABT-888. Using a proliferative assay, myeloma cells appeared sensitive to ABT-888 with low GI50 values (8.7μM for RPMI8226 cells, 49μM for U266 cells) and increased γH2AX foci, which persisted at 24 hours after treatment. This was confirmed in methycellulose colony assay where ABT-888 treatment reduced RPMI8226 colonies by 35% (p=0.002). Next we showed synergistic cytotoxicity between ABT-888 and melphalan. In both RPMI8226 and U266 cells strong synergy was displayed with a combination index (CI) less than 1 in proliferative assays (CI 0.5 and 0.3 at 50% proliferation respectively). Combination ABT-888 and melphalan treated cells underwent accelerated senescence compared to cells treated by melphalan alone (27% versus 51% βGal+ staining at 24 hours, p=0.02). This was confirmed by upregulation of senescence related genes p16 (1.6 fold increase) and p21 (1.5 fold increase). We did not find significant difference in apoptosis by Annexin V/ PI staining. Given that increased non-homologous end joining (NHEJ) activity has been shown to lead to resistance to melphalan, we tested whether an inhibitor of NHEJ could be synergistic with PARP inhibition and melphalan. Treatment with the DNA-PK inhibitor NU7026 at 10μM in addition to ABT-888 at 4μM resulted in 46% reduction in proliferation in RPMI8226 cells and 52% in U266 cells. When used in combination with melphalan chemotherapy, the dual DNA repair inhibitor therapy showed marked synergy in RPMI8226 cells with a combination index of 0.39. Finally we tested the ability of the combination of ABT-888 and melphalan to treat myeloma in-vivo. NSG mice were injected via tail vein with 5x106 RPMI8226 cells. Control (untreated) mice subsequently developed myeloma infiltrating the marrow, spleen and axial skeleton, with hind limb paralysis occurring at a median of 42 days. Treated mice received intraperitoneal injections of ABT-888 (3 times a week), or melphalan (weekly) or a combination of both agents starting on day 28 post-injection of MM cells for a total of 3 weeks. Using ABT-888, melphalan and a combination of both agents, median survival of mice was progressively prolonged (44 vs. 67 vs. 107 days, respectively) (p=0.02).Here we show that PARP and DNA-PK inhibition synergizes with melphalan in myeloma cells lines, providing a rationale for the addition of these agents to conditioning chemotherapy. In addition, we also show a direct anti-myeloma activity of these agents without the use of alkylator chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Study of the effects in vitro of berberine combined with bortezomib on multiple myeloma cell line U266 cells

目的探讨小檗碱与硼替佐米联合对多发性骨髓瘤（MM）细胞增殖及诱导细胞凋亡的作用及其可能的机制。方法以MM细胞株U266细胞为研究对象，以小檗碱和硼替佐米作用后，采用MTT法检测其对U266细胞增殖的影响，采用流式细胞术检测其对U266细胞凋亡的影响，采用ELISA法检测其对细胞caspase-3、-8、-9表达的影响，采用Western blot法检测其对细胞Fas相关死亡结构域蛋白（FADD）、TNF受体相关死亡域蛋白（TRADD）表达的影响。采用金（正均）氏公式计算协同系数Q值分析两药的协同作用。结果①两药单独作用时对U266细胞增殖的抑制作用均呈时间、剂量依赖关系；小檗碱（20 µmol/L）与硼替佐米（5 nmol/L）联合作用对U266细胞增殖具有协同抑制作用（Q值为1.31~1.65）。②无论单独用药还是联合用药，与对照组比较，用药组早期凋亡细胞的比例均显著增加（P值均<0.05）；两药联合对U266细胞具有协同促进凋亡的作用（作用6、12 h, Q值分别为0.896和1.197）。③两药联合应用后U266细胞caspase-3、-8、-9、表达水平均较对照组、单药组明显增加，差异有统计学意义（P值均<0.05）。④两药联合应用（24、48 h）后U266细胞TRADD（0.91±0.01、0.70±0.01）、FADD（0.98±0.01、0.98±0.01）蛋白水平均较对照组（0.54±0.01、0.41±0.01）明显增加，差异有统计学意义（P值均<0.05）。结论小檗碱联合硼替佐米可协同促进MM细胞凋亡，这种作用可能是通过上调TRADD及FADD蛋白表达水平而实现的。

Effect of dexamethasone combined with oridonin on proliferation and apoptosis of multiple myeloma cell line U266

Objective To investigate the effect of dexamethasone combined with oridonin on proliferation and apoptosis in multiple myeloma cells U266 and the related molecular mechanism. Methods Exponential phase of growth U266 cells were treated with different concentrations of oridonin combined with dexamethasone or alone. U266 cells treated by DMSO were taken as control group. The proliferation inhibitory ratios were measured by CCK-8 assay followed by 24 h, 48 h and 72 h. Apoptosis induction was assessed by using Annexin V-FITC kit. Real time PCR was used to examine the mRNA changes of Notch1, NF-κB/p65 and bcl-2. Western blot assay was applied to detect the protein expression of Notch1, cleaved Notch1, NF-κB/p65 and bcl-2. Results Compared with that in control group, proliferation in all the experimental groups was inhibited(P<0.05), and the apoptosis was promoted(P<0.05); especially the combination of dexamethasone and oridonin had a synergistic effect on the proliferation and apoptosis of U266 cells(P<0.05). The results of PCR and Western blot showed that after treatment of U266 cells with dexamethasone, the mRNA as well as their protein levels of NF-κB/p65 and bcl-2 were decreased compared with those in the control group(P<0.05). Moreover, the mRNA and protein expression of Notch1, cleaved Notch1, NF-κB/p65 and bcl-2 was obviously down-regulated in oridonin group and the combination group(P<0.05). Conclusion Combination of dexamethasone and oridonin can significantly increase the anti-tumor effect by inhibiting proliferation and inducing apoptosis of U266 cells, which may be related to the inhibition of the Notch1 pathway. Key words: Multiple myeloma; Oridonin; Dexamethasone; Proliferation; Apoptosis

Increase of CIK cell efficacy by upregulating cell surface MICA and inhibition of NKG2D ligand shedding in multiple myeloma.

Multiple myeloma, which is a monoclonal plasma cell malignancy, still remains incurable despite recent progress in our understanding of this disorder. Adoptive immunotherapy of multiple myeloma using cytokine-induced killer cells is yielding promising results in clinical trials; however, some myeloma cells still evade immune surveillance by various unknown molecular mechanisms. This study aims at increasing the efficacy of cytokine-induced killer cells in targeting this tumor, using selective small-molecule inhibitors which increase and stabilize surface expression of the natural killer group 2, member D ligand, major histocompatibility complex class I polypeptide-related sequence A (MICA) on myeloma cells. We treated 2 multiple myeloma cell lines-U266 and KMS-12-PE-with 3 drugs. One of these drugs (sodium butyrate) is a histone deacetylase inhibitor. Another drug which was used (matrix metalloproteinase inhibitor III) blocks ligand shedding while the third drug (phenylarsine oxide) obstructs surface ligand internalization. The effect of these drugs on cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, surface ligand expression was examined using flow cytometry, and ligand shedding was assessed using enzyme-linked immunosorbent assay. We demonstrated that cytokine-induced killer cells have increased cytotoxicity against multiple myeloma cells after combined drug treatment than without drug pretreatment. We also established that this increased cytotoxicity was due to potent upregulation and stabilization of surface MICA on the surface of these tumor cell lines. Our study thus highlights further therapeutic options which could be used for the treatment of multiple myeloma patients.

Valproic acid activates autophagy in multiple myeloma cell lines RPMI8226 and U266

目的探讨丙戊酸钠对多发性骨髓瘤（MM）细胞株RPMI8226和U266细胞自噬的影响。方法丙戊酸钠处理RPMI8226和U266细胞，吖啶橙染色后采用荧光显微镜观察细胞自噬形态学变化，MTT法检测细胞增殖抑制的变化，流式细胞术检测细胞凋亡，实时定量PCR（RT-PCR）和Western Blot法检测细胞自噬相关因子LC3、Beclin1的变化。结果荧光显微镜观察到RPMI8226及U266细胞存在基础水平的自噬现象，丙戊酸钠作用后能够诱导细胞自噬增多；MTT法检测结果显示丙戊酸钠对细胞增殖抑制具有时间及浓度依赖性，作用24 h后半数抑制浓度分别为（12.03±0.23）mmol/L和（10.16±0.37) mmol/L。8 mmol/L丙戊酸钠作用24 h后，RPMI8226、U266细胞LC3 mRNA表达水平（22.45±0.07、0.06±0.02）、Beclin1 mRNA表达水平（283.09±17.3、1.53±0.01）与空白对照组（1.00± 0.00、1.00±0.00）比较，差异均有统计学意义（P值均<0.05）。随着丙戊酸钠浓度增加和作用时间延长，LC3、Beclin1蛋白表达水平逐渐增加，LC3Ⅰ向LC3Ⅱ的转化率逐渐升高。结论RPMI8226和U266细胞中存在基础水平的自噬现象，丙戊酸钠对MM细胞的自噬有激活作用，这可能是丙戊酸钠治疗MM的机制之一。

Silybin suppresses cell proliferation and induces apoptosis of multiple myeloma cells via the PI3K/Akt/mTOR signaling pathway

Silybin is a biologically active component extracted from the seeds of Silybum marianum, which has been shown to have inhibitory effects on prostate, skin, bladder, lung and colon cancer cells, in addition to its efficacy in the treatment of liver diseases, including hepatitis and cirrhosis. The aim of the present study was to investigate whether silybin suppresses the proliferation and induces apoptosis of multiple myeloma (MM) cells and to elucidate its molecular targets. The proliferative and apoptotic rates of the U266 MM cell line were assessed using MTT and flow‑cytometric assays, respectively. Western blot analysis was used to assess the protein levels of phosphoinositide‑3 kinase (PI3K), phosphorylated (p)‑Akt and p‑mammalian target of rapamycin (mTOR) in U266 cells. In addition, PI3K inhibitor LY294002 or activator insulin‑like growth factor 1 were used to investigate the involvement of the PI3K/Akt‑mTOR signaling pathway in the effect of silybin on U266 cells. The results revealed that silybin restrained the proliferation and enhanced the apoptosis of U266 cells. Furthermore, silybin inhibited the protein expression of PI3K, p‑Akt and p‑mTOR in U266 cells. Of note, inhibition of PI3K facilitated silybin‑mediated reduction of mTOR activation, cell proliferation and induction of apoptosis in U266 cells, while activation of PI3K attenuated the effects of silybin. In conclusion, silybin suppressed cell proliferation and promoted apoptosis of U266 cells via PI3K/Akt-mTOR signaling pathways.

T Cell Receptor Gene Therapy Targeting the Intracellular Transcription Factor Bob1 for the Treatment of Multiple Myeloma and Other B Cell Malignancies

Therapeutic reactivity of CD20-specific monoclonal antibodies (mAb) or CD19-specific chimeric antigen receptor (CAR)-transduced T cells is exerted by targeting extracellular antigens. In contrast to mAbs and CARs, T cell receptors (TCRs) recognize antigen-derived peptides that are bound to human leukocyte antigen (HLA) molecules on the cell surface. Since HLA molecules constantly sample the entire endogenous proteome of a cell, extracellular and intracellular antigens are presented and can thus be recognized by a TCR.Here, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for immunotherapy. Bob1 is highly expressed in CD19+ B cells, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and multiple myeloma (MM) and is absent in the non-B lineages including CD34+ hematopoietic progenitor cells (HPCs), T cells, fibroblasts, keratinocytes and gastrointestinal tract. Bob1 is localized intracellularly but HLA-presented Bob1-derived peptides are accessible on the cell surface to TCRs and can thus be recognized by T cells. From the HLA-presented ligandome (Mol Cell Proteomics, 2013;12:1829) we identified naturally processed Bob1-derived peptides displayed in HLA-A*0201 (HLA-A2) and in HLA-B*0702 (HLA-B7). Since auto-reactivity towards self-antigens such as Bob1 is prevented by depleting high-avidity T cells recognizing self-antigens in self-HLA, we exploited the immunogenicity of these peptides presented in allogeneic HLA. From a HLA-A2/B7-negative healthy individual we isolated T cell clone 4G11 demonstrating high sensitivity and specificity for Bob1-derived peptide Bob144 presented in HLA-B7. Bob1-dependent recognition was demonstrated by transduction of Bob1 into cell lines that otherwise lack Bob1 expression. No harmful toxicities of clone 4G11 were observed against a wide panel of Bob1-negative stimulator cells including HLA-B7-positive CD34+ HPCs, T cells, monocytes, immature and mature dendritic cells, and fibroblasts even under simulated inflamed conditions. Furthermore, stringent HLA-B7-restricted recognition was observed for clone 4G11 when tested against a stimulator panel expressing a wide range of common and rare HLA class I and II molecules. Clone 4G11 demonstrated clinical applicability by efficiently recognizing HLA-B7+ primary ALL, CLL and MCL. Furthermore, reproducible strong recognition of purified primary HLA-B7+ MM could be demonstrated.Therefore, the TCR of clone 4G11 may be used for immunotherapy by administering TCR-transduced T cells to patients suffering from B cell malignancies including multiple myeloma. Retroviral gene transfer of TCR 4G11 led to efficient cell surface expression demonstrated by binding of TCR-transduced CD8+ T cells to pMHC-tetramer composed of peptide Bob144 bound to HLA-B7. TCR-modified CD8+ T cells strongly recognized Bob1-expressing HLA-B7+ multiple myeloma cell lines U266 and UM9, and ALL cell lines. TCR-modified T cells efficiently lysed HLA-B7+ primary ALL, CLL and MCL at very low effector-to-target ratios. In addition, highly purified primary multiple myeloma samples were also readily lysed. Furthermore, TCR-transduced T cells strongly proliferated in an antigen-specific manner when stimulated with primary malignant cell samples including ALL, CLL, and MCL or MM cell lines. As expected, TCR-transduced T cells also lysed autologous primary and CD40L-stimulated B cells since these targets cells also express Bob1. In contrast, no lysis of Bob1-negative autologous primary and activated T cells, or monocytes was observed when co-cultured with TCR-transduced T cells.In summary, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for TCR-based immunotherapies of B cell malignancies. Bob1-specific T cell clone 4G11 efficiently recognized primary B cell leukemia and multiple myeloma. Gene transfer of TCR of clone 4G11 installed Bob1-reactivity and specificity onto recipient T cells shown here by cytolytic capacity and proliferation upon antigen encounter. TCR gene transfer approaches using this Bob1-specific TCR can bring novel treatment modalities and possibly curative therapy to patients with B cell malignancies including multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

5-Azacitidine Remolds the Methylation Status and Inhibits Growth in Multiple Myeloma

Multiple myeloma (MM) is a malignant disorder characterized by the proliferation of a single clone of plasma cells derived from B cells. Previous studies have demonstrated that both gene-specific hypermethylation and global hypomethylation characterizes the multiple myeloma epigenome. 5-azacytidine as a DNA methylation inhibitor has therapeutic efficacy in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Nevertheless,the effects of 5-azacytidine on MM remains unclear. We used RT-PCR to detect the expression of PTPL1 and used MS-PCR to determine the methylation status of PTPL1 in MM cell lines and after 5-azacytidine treatment. ELISA-like reaction was used to detect global DNA methylation level. The cytotoxic activity of 5-azacytidine was tested using cell viability and apoptosis assays. Flow cytometry was used to detect cell cycle after 5-azacytidine treatment. Our experiments discovered that the PTPL1 gene was hypermethylated in the U266 and H929 cell lines, and the expression of PTPL1 mRNA could be re-inducible by 5-azacytidine. 5-azacytidine also inhibited the proliferation of multiple myeloma cell lines U266 and H929 in a time- and dose-dependent manner, induced G2/M cell cycle arrest and caspase-dependent apoptosis. But in our study 5-azacytidine increased the methylation level for both cell lines. Our study showed that PTPL1 was epigenetically regulated in MM which can be reversed by 5-azacytidine, and highlights 5-azacytidine is a potential therapeutic candidate for MM, but additional studies are needed to determine the effects of genome-wide methylation changes in MM. DisclosuresNo relevant conflicts of interest to declare.

A New Class of T-Cell Bispecific Antibodies for the Treatment of Multiple Myeloma, Binding to B Cell Maturation Antigen and CD3 and Showing Potent, Specific Antitumor Activity in Myeloma Cells and Long Duration of Action in Cynomolgus Monkeys

A New Class of T-Cell Bispecific Antibodies for the Treatment of Multiple Myeloma, Binding to B Cell Maturation Antigen and CD3 and Showing Potent, Specific Antitumor Activity in Myeloma Cells and Long Duration of Action in Cynomolgus Monkeys

Stem Cell-like Characteristics of MM Plasma Cells Vary By ROS Levels: Implications for Targeted Therapy

Background: Tumor cells exhibit elevated ROS levels and cancer stem cells have lower ROS levels than their normal counterparts [Kobayashi et al., 2011]. However, little is known about the effect of ROS levels on multiple myeloma (MM) plasma cell (PC) chemosensitivity. Previously we found that ROS levels are variable in CD138-selected MMPCs from different cases at diagnosis. However, ROS levels in MMPCs from focal lesions (FL, n=42) are lower than PCs from random bone marrow (BM) aspirates (n=192, p=0.02). ROS levels also vary among MM molecular subgroups, with MF and CD2 subgroups having low ROS levels, suggesting that the microenvironment and genetic factors affect their oxidative capacities. We hypothesized that ROS level may reflect the cancer stemness properties of MM cells. We first sought to establish a model to investigate MM cells with different ROS levels and then determine if this property confers cancer stemness and chemoresistance.Methods: We analyzed gene expression profiles (GEP) of primary myeloma cells from newly diagnosed patients with low (n=48) and high (n=48) ROS level by gene set enrichment analysis. We compared viability, proliferation, cell cycle status, drug efflux ability, chemosensitivity, and clonogenic capacity of MM cell lines H929 and U266 with high and low ROS levels, sorted by FACS using H2DCFDA. We also investigated whether MMPCs with low or high ROS levels would differ in their sensitivity to BBI-608, a drug that is reported to target STAT3 driven gene transcription and cancer stemness properties in solid cancers [Li et al., 2015] and is currently being tested in hematologic cancers [BBI608-103HEME].Results: Pathways enrichment analysis revealed that primary MM cases with low ROS had increased oxidative phosphorylation, proteasome activity, and nucleotide excision repair compared to cases with high ROS that had increased glycolysis, and chemokine and calcium signaling pathways. To explore these differences we separated MMCLs into cells not sorted by ROS level (non-ROS sorted), ROSLo, and ROSHi groups. After sorting, H929 cells retained significant differences in ROS levels for 48-72 hr and U266 cells returned to basal ROS levels within 24hr. ROSLo H929 cells had significantly reduced cell number (p<0.0001) compared to non-ROS sorted and ROSHi cells. Similarly, ROSLo cells had reduced and ROSHi cells had significantly increased proliferation index compared to non-ROS sorted cells (H929-p<0.05, U266-p<0.001). ROSLo cells also have a significantly larger percentage of cells in S phase (p <0.01) and reduced percentage in G2 (p< 0.001) compared to non-ROS sorted cells. While ROSHi cells (but not ROSLo cells) were less sensitive to melphalan compared to non-ROS sorted cells, there was no difference in sensitivity to bortezomib. Examination of ABC transporter efflux activity revealed that ROSHi cells had increased drug efflux capacity via the multidrug resistance protein MRP1 (p<0.01). ROSLo cells showed greater colony forming capacity, however after serial replating, ROSHi cells increase colony forming capacity after each passage eventually matching that of ROSLo cells. Interestingly, if clonogenic cells are re-sorted into ROSLo and ROSHi groups between passages, ROSLo cells retain superior colony forming capacity compared to ROSHi cells. ROSLo (IC50-0.656μM) and ROSHi (IC50-0.56μM) cells had significantly increased sensitivity to BBI-608 compared to non-ROS-sorted cells (IC50-0.991 μM). Cell number was significantly reduced in all 3 groups without significantly affecting viability. The STAT3 target gene, CCND1 is significantly elevated in ROSHi cells and effectively reduced by BBI-608. After low dose BBI-608 (0.6μM) treatment, the remaining cells were subjected to colony forming assays. While BBI-608 reduced colony forming capacity to some extent in all 3 groups, ROSHi cells were most sensitive.Conclusions: Characterization of MM cells with variable ROS levels revealed that ROSHi cells have greater drug efflux capacity and are more proliferative and chemoresistant, whereas ROSLo cells have reduced proliferation and increased clonogenic capacity. ROSLo MMPCs may represent a subpopulation of tumor cells with stemness properties that are enriched in the hypoxic BM niche and in MM focal lesions. DisclosuresMalaviarachchi:University of Arkansas for Medical Sciences: Employment. Turner:University of Arkansas for Medical Sciences: Employment. Stein:University of Arkansas for Medical Sciences: Employment. Barlogie:University of Arkansas for Medical Sciences: Employment. Morgan:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; University of Arkansas for Medical Sciences: Employment; CancerNet: Honoraria; Weismann Institute: Honoraria. Epstein:University of Arkansas for Medical Sciences: Employment. Yaccoby:University of Arkansas for Medical Sciences: Employment. Johnson:University of Arkansas for Medical Sciences: Employment.

Mechanisms of Apoptosis Induced by FTY720 in Multiple Myeloma Cell Line U266

To investigate the effects of FTY720 on apoptosis in multiple myeloma cell line U266 and to clarify the molecular mechanism of apoptosis induced by FTY720. U266 cells were treated with 2.5, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the apoptotic rates were tested by flow cytometry with Annexin-V-FITC/PI staining. Then U266 cells were treated with 20 µmol/L FTY720 for 0, 6, 16 and 24 hours, the apoptotic rates were tested. U266 cells were treated with DMSO and FTY720 separately and then were stained with DAPI for 5 min. Drop the cells to the slides and cover the slide with the glass. The cells were observed by fluorescence microscopy. U266 cells were treated with 5 µmol/L FTY720 or together with different doses of Z-VAD-fmk (12.5, 25, 50 µmol/L), a pancaspase inhibitor, for 24 hours, then the cell viability was tested by CCK-8. U266 cells were treated with 2.5, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the expression of cleaved caspase-3 was tested by Western blot. U266 cells were treated with 0, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the expressions of MCL-1, survivin, BCL-2, BID, BAX, BAK, P-ERK were tested by Western blot. The apoptotic rate increased in U266 cells treated with FTY720 and showed the characteristic of time-dependent and dose-dependent manner. Karyopyknosis and nuclearfragmentation could be observed in U266 cells treated with FTY720 after being stained with DAPI under fluorescent microscope. The same effect was not observed in the cells treated with DMSO. Z-VAD-fmk could rescue the apoptosis in U266 cells treated with FTY720 in dose-dependent manner. The expression of MCL-1, survivin and BCL-2 decreased in U266 cells treated with FTY720. The cleavage of BID could be observed in U266 cells treated with FTY720. FTY720 had no effect on the expression of BAX, BAK and P-ERK. FTY720 can induce the apoptosis in U266 cells, the apoptosis was Caspase-3-depended. The apoptosis induced by FTY720 is due to the decrease of MCL-1, survivin and BCL-2, which are the inhibitors of apoptosis. Meanwhile, the apoptosis was also due to the activation of BID, which is pro-apoptotic protein.

Histone deacetylase inhibitor LBH589 induces apoptosis of multiple myeloma cell line U266 in vitro and its mechanism

Objective To study the effect of new generation histone deacetylase inhibitor LBH589 single-drug or combined with the mouse serum which contains the radix echinopsis on multiple myeloma (MM) cell line U266 and their mechanism. Methods The acetylation level of α-tublin which was a substrate of HDAC6 was detected by Western blot.The chemical force between Heat shock protein 90 (HSP90) and its client proteins was detected by immune precipitation (IP). Results The different concentrations of LBH589 single drug (0, 20, 50 nmol/L), and 50 nmol/L combined with the mouse serum which contained the radix echinopsis (1 g/ml) were able to inhibit the proliferation of U266 cell. With the increase of drug concentration and the extension of time, the acetylation levels of α-tublin and HSP90 increased gradually (at 24 or 48 hours) in a dose dependent (P < 0.05). The inhibition of LBH589 combined with the mouse serum was stronger than that of LBH589 single drug (P < 0.05). Conclusion LBH589 could inhibit the growth of MM cells and their cell cycles, and induce the apoptosis of MM cell line U266. Key words: Multiple myeloma; LBH589; Western blot; Immune precipitation; Heat shock protein 90; α-tubulin

Study of NK cells dysfunction in multiple myeloma patients

目的探讨多发性骨髓瘤（MM）患者NK细胞杀伤功能下降的机制。方法以13例MM患者为研究对象，以30名健康志愿者为正常对照，采用流式细胞术检测外周血单个核细胞中CD3 −CD56+（NK细胞）表面抑制性受体（CD158a和CD158b）以及活化性受体NKG2D、NCRs（NKp30、NKp44、NKp46）的表达水平；ELISA法检测血清可溶性NKG2D配体水平；流式细胞术检测NK细胞对MM细胞株U266细胞杀伤作用的变化。结果MM患者外周血中NK细胞比例及绝对数、CD158a和CD158b表达水平与对照组比较差异无统计学意义（P值均>0.05），两组NK细胞表面均不表达NKp44；与对照组比较，MM患者NK细胞表面活化性受体（NKG2D、NKp30和NKp46）水平均明显降低，血清中可溶性NKG2D配体水平明显升高，差异均有统计学意义（P值均<0.05）；用MM患者血清培养后的正常对照者NK细胞对U266细胞的杀伤能力较共培养前明显下降[（25.4±5.9）％对（38.5±6.5）%]，差异有统计学意义（P=0.044）。结论MM患者血清可溶性NKG2D配体水平可能是导致NK细胞杀伤活性下降的原因。

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.

Evaluation of the change in sphingolipids in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 treated with arsenic trioxide.

Arsenic trioxide (As2O3) has been found to display anticancer activity against many types of tumors and has been developed into an anticancer drug in clinical treatments. Sphingolipids are membrane lipids that participate in many signal transduction pathways. In this paper, the changes in sphingolipids of the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803 treated with arsenic trioxide were investigated using an HPLC-ESI-MS/MS method. Analytes were separated by an XBridge BEH C8 column used for Cer, HexCer, LacCer and SM chromatographic separation, and a Capcell PAK MG II C18 column was used for Sph, dhSph, S1P and dhS1P chromatographic separation and gradient elution with acetonitrile-water containing 0.1% formic acid as a mobile phase. A tandem mass spectrometer QTrap in SRM mode was employed in combination with RPLC as a detector for quantitative analysis. The ceramide/sphingolipid internal standard (IS) mixture was used to quantify the levels of sphingolipids. The distributions of sphingolipids were found to be different in the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803. Ceramide (Cer), hexosylceramide (HexCer) and dihexosylceramide (Hex2Cer) levels in U266 cell line are higher than those in MGC-803 cell line. Additionally, sphingomyelin (SM), sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (dhS1P) levels in the MGC-803 cell line are higher than those in the U266 cell line. When treated with arsenic trioxide (1-5μM iAs(III)(As(III) ions)), the levels of Hex2Cer in the human multiple myeloma cell line U266 decreased, and the levels of S1P and dhS1P in the human gastric cancer cell line MGC-803 decreased. The decrease of Hex2Cer, S1P and dhS1P in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 were observed when the concentration of iAs(III) is 1.0μM. Therefore, arsenic trioxide exhibits anti-cancer activity by altering the sphingolipid pathway in the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803.