Bortezomib Inhibits Multiple Myeloma Cells by Transactivating ATF3 to Trigger miR-135a-5p- Dependent Apoptosis

Multiple myeloma (MM) is a malignant cancer with an increasing in incidence that can be alleviated through bortezomib (BTZ) treatment. Activating transcription factor 3 (ATF3) plays a major role in cancer development. Moreover, microRNAs (miRNAs) regulate carcinogenic pathways, apoptosis, and programmed necrotic cell death. However, the detailed mechanism by which ATF3 modulates BTZ drug sensitivity/resistance remains elusive. In the current study, expression of ATF3 was significantly increased under BTZ treatment in a dose-dependent manner in MM cell lines. In addition, ATF3 could regulate cell apoptosis under BTZ treatment. The effect of ATF3 was negatively regulated by its binding miRNA, miR-135a-5p. When either ATF3 was silenced or miR-135a-5p mimics were added to MM cells, they partially lost sensitivity to BTZ treatment. This was accompanied by low levels of Noxa, CHOP, and DR5, and a decrease in mitochondrial membrane potential. These results revealed the combinatorial regulatory patterns of ATF3 and miR-135a-5p in the regulatory protein interactome, which indicated a clinical significance of the miR-135a-5p-ATF3 protein interaction network in BTZ therapy. This study provides potential evidence for further investigation into BTZ resistance.

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Steroidogenic Factor 1 (NR5A1) Activates ATF3 Transcriptional Activity

International Journal of Molecular Sciences ◽

10.3390/ijms21041429 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1429

Author(s):

Natsuko Emura ◽

Chiung-Min Wang ◽

William Harry Yang ◽

Wei-Hsiung Yang

Keyword(s):

Site Directed Mutagenesis ◽

Activating Transcription Factor 3 ◽

Dependent Manner ◽

Orphan Nuclear Receptor ◽

Steroidogenic Factor 1 ◽

Post Translational Modifications ◽

Endocrine Organs ◽

Activating Transcription Factor ◽

Dose Dependent ◽

Atf3 Expression

Steroidogenic Factor 1 (SF-1/NR5A1), an orphan nuclear receptor, is important for sexual differentiation and the development of multiple endocrine organs, as well as cell proliferation in cancer cells. Activating transcription factor 3 (ATF3) is a transcriptional repressor, and its expression is rapidly induced by DNA damage and oncogenic stimuli. Since both NR5A1 and ATF3 can regulate and cooperate with several transcription factors, we hypothesized that NR5A1 may interact with ATF3 and plays a functional role in cancer development. First, we found that NR5A1 physically interacts with ATF3. We further demonstrated that ATF3 expression is up-regulated by NR5A1. Moreover, the promoter activity of the ATF3 is activated by NR5A1 in a dose-dependent manner in several cell lines. By mapping the ATF3 promoter as well as the site-directed mutagenesis analysis, we provide evidence that NR5A1 response elements (−695 bp and −665 bp) are required for ATF3 expression by NR5A1. It is well known that the transcriptional activities of NR5A1 are modulated by post-translational modifications, such as small ubiquitin-related modifier (SUMO) modification and phosphorylation. Notably, we found that both SUMOylation and phosphorylation of NR5A1 play roles, at least in part, for NR5A1-mediated ATF3 expression. Overall, our results provide the first evidence of a novel relationship between NR5A1 and ATF3.

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Anti-tumor activity of a novel proteasome inhibitor D395 against multiple myeloma and its lower cardiotoxicity compared with carfilzomib

Cell Death and Disease ◽

10.1038/s41419-021-03701-z ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Xuxing Shen ◽

Chao Wu ◽

Meng Lei ◽

Qing Yan ◽

Haoyang Zhang ◽

...

Keyword(s):

Multiple Myeloma ◽

Proteasome Inhibitor ◽

Osteoclast Differentiation ◽

Dependent Manner ◽

Serum Enzyme ◽

Herg Channels ◽

Anti Tumor Activity ◽

Dose Dependent

AbstractCarfilzomib, a second-generation proteasome inhibitor, has significantly improved the survival rate of multiple myeloma (MM) patients, but its clinical application is still restricted by drug resistance and cardiotoxicity. Here, we identified a novel proteasome inhibitor, D395, and assessed its efficacy in treating MM as well as its cardiotoxicity at the preclinical level. The activities of purified and intracellular proteasomes were measured to determine the effect of D395 on the proteasome. CCK-8 and flow cytometry experiments were designed to evaluate the effects of D395 on cell growth and apoptosis. The effects of D395 and carfilzomib on serum enzyme activity, echocardiography features, cardiomyocyte morphology, and hERG channels were also compared. In our study, D395 was highly cytotoxic to MM cell lines and primary MM cells but not normal cells, and it was well tolerated in vivo. Similar to carfilzomib, D395 inhibited osteoclast differentiation in a dose-dependent manner. In particular, D395 exhibited lower cardiotoxicity than carfilzomib in all experiments. In conclusion, D395 is a novel irreversible proteasome inhibitor that has remarkable anti-MM activity and mild cardiotoxicity in vitro and in vivo.

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A Fully Human Antagonist Anti-CD40 Antibody Triggers Significant Antitumor Activity Against Human Multiple Myeloma.

Blood ◽

10.1182/blood.v104.11.2414.2414 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2414-2414

Author(s):

Yu-Tzu Tai ◽

Xian-Feng Li ◽

Xia Tong2 ◽

Laurence Catley ◽

Daniel Santos ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Dependent Manner ◽

Peripheral Blood Mononuclear ◽

Specific Lysis ◽

Growth And Survival ◽

Human Multiple Myeloma ◽

Dose Dependent

Abstract We previously demonstrated that CHIR-12.12, a fully human anti-CD40 mAb (IgG1) generated in XenoMouseÒ mice (Abgenix, Inc), blocks CD40/CD40 ligand (CD40L) interactions and has more potent anti-lymphoma activity than Rituximab both in vivo and in vitro (abstract #2386, ASH, San Diego, Dec. 2003). In this study, we assess the efficacy of CHIR-12.12 against human multiple myeloma (MM) using CD40-expressing MM cell lines and purified CD138+ patient cells. CHIR-12.12 binds to purified CD138+ MM cells in >80% (10/12) of patient samples, as measured by flow cytometry: the mean fluorescence intensity (MFI) range was 1 to 20 for CHIR-12.12 vs 0.2–0.9 for control human IgG1. We next examined the antagonist activity of CHIR-12.12 in MM cells. CHIR-12.12 blocked CD40L-mediated proliferation of CD40-expressing MM lines and purified CD138+ patient cells from 2 MM patients in a dose-response manner. In contrast, CHIR-12.12 alone did not alter constitutive MM cell proliferation. Immunoblotting analysis demonstrated that PI3-K/AKT, NF-kB, and ERK activation induced by hCD40L in the 12BM MM cell line was significantly inhibited by CHIR-12.12 (5 μg/ml). Adhesion of MM cells to bone marrow stromal cells (BMSCs) confers growth and survival benefit for tumor cells. Since CD40 activation, either by stimulatory mouse anti-CD40 mAb G28.5 or formaldehyde-fixed CHO cells expressing hCD40L, induces MM cell adhesion to fibronectin (FN) or BMSCs, we next asked whether antagonist CHI12.12 abrogates this process. CHIR-12.12 inhibited CD40L-induced adhesion of MM cell lines to FN in a dose dependent manner (0.001-10 μg/ml), whereas control human IgG did not. Moreover, CHIR-12.12 (1 μg/ml) blocked hCD40L-induced adhesion of freshly isolated patient MM cells to BMSCs. Adhesion of MM cells to BMSCs induces IL-6 secretion, an important growth and survival cytokine for MM cells, and treatment of MM cells with hCD40L further augmented adhesion-induced IL-6 secretion. Conversely, pretreatment of CD40-expressing MM cell lines with CHIR-12.12 significantly decreased IL-6 secretion triggered by coculture of MM cells with BMSCs. We next examined whether CHIR-12.12 stimulates antibody-dependent cellular cytotoxicity (ADCC) against CD40-expressing MM cells. Human peripheral blood mononuclear cells and purified NK cells (CD56+CD3−) were used as effector cells. CHIR-12.12 triggered MM cell lysis in a dose dependent manner, as measured in CD40-expressing MM cell lines. The maximum specific lysis of 20–70 % was achieved at 10 μg/ml concentration of CHIR-12.12. CHIR-12.12 mediated lysis was specific to CD40-expressing MM cells, as CHIR-12.12 did not induce ADCC against CD40-negative MM cells. Importantly, CHIR-12.12 induced ADCC against CD138+ cells isolated from 2 MM patients. These results provide preclinical rationale for clinical evaluation of CHIR-12.12 with the goal of improving patient outcome in MM.

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The New CXCR4 Inhibitor MDX-1338 Exerts Anti-Tumor Activity in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.1844.1844 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1844-1844 ◽

Cited By ~ 1

Author(s):

Aldo M Roccaro ◽

Antonio Sacco ◽

Michelle Kuhne ◽

AbdelKareem Azab ◽

Patricia Maiso ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cells ◽

Mouse Models ◽

Research Funding ◽

Therapeutic Agents ◽

Dependent Manner ◽

Apoptotic Pathways ◽

Dose Dependent

Abstract Abstract 1844 Background. We have previously shown the SDF1/CXCR4 axis plays a major role in homing and trafficking of multiple myeloma (MM) to the bone marrow (BM), and disruption of the interaction of tumor cells with the BM leads to enhanced sensitivity to therapeutic agents. We hypothesize that the novel anti-CXCR4 antibody, BMS936564/MDX-1338, may prevent the homing and adhesion of MM cells to the BM and will sensitize them to therapeutic agents. Methods. Primary MM cells (CD138+); MM cell lines (MM.1S, RPMI.8226); and primary MM bone marrow stromal cells (BMSCs) were used. Migration towards SDF-1 and BMSCs has been evaluated. Cytotoxicity and DNA synthesis were measured by MTT and thymidine uptake, respectively. Cell signaling and apoptotic pathways were studied by Western Blot. Synergism was calculated using the Chou-Talalay method. In vivo MM tumor growth was evaluated with xenograft mouse models. Results. MDX-1338 inhibited migration of MM cells toward SDF-1a and primary MM BMSCs, in a dose-dependent manner. Adhesion of primary MM cells to BMSCs was also inhibited by BMS936564/MDX-1338 in a dose-dependent manner, while also inducing cytotoxicity on primary BM-derived CD138+ cells. BMS936564/MDX-1338 targeted MM cells in the context of BM milieu by overcoming BMSC-induced proliferation of tumor cells. In addition, BMS936564/MDX-1338 synergistically enhanced bortezomib-induced cytotoxicity in MM cells. BMS936564/MDX-1338-dependent activation of apoptotic pathways in MM cells was documented, as shown by cleavage of caspase-9 and PARP. SDF-1a-induced ERK-, Akt-, and Src-phosphorilation was inhibited by BMS936564/MDX-1338 in a dose-dependent manner. Importantly, BMS936564/MDX-1338 inhibited MM cell proliferation in vivo in xenograft mouse models. Conclusion. These studies therefore show that targeting CXCR-4 in MM by using BMS936564/MDX-1338 represents a valid therapeutic strategy in this disease. Disclosures: Roccaro: Roche:. Kuhne:BMS: Employment. Pan:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Ghobrial:Noxxon: Research Funding; Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celegene:; Novartis:.

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Multikinase Inhibitor Sorafenib Induces Apoptosis, CD138-Downregulation, Actin Depolymerization and Inhibition of M210B4-Triggered Chemotaxis in Multiple Myeloma Cell Lines and Synergyzes with Proteasome Inhibitor Bortezomib

Blood ◽

10.1182/blood.v118.21.2380.2380 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2380-2380

Author(s):

Josefina Udi ◽

Dagmar Wider ◽

Julie Catusse ◽

Dominik Schnerch ◽

Marie Follo ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Proliferation ◽

Actin Polymerization ◽

Combination Index ◽

Dependent Manner ◽

Multikinase Inhibitor ◽

And Migration ◽

Dose Dependent ◽

Morphologic Changes

Abstract Abstract 2380 Introduction: Sorafenib is an oral multikinase inhibitor that targets several cancer-specific pathways and directly affects tumor cell proliferation, cell survival and neovascularization. The Ras/Raf/MEK/ERK pathway is particularly known to be critical for proliferation of multiple myeloma (MM) cells. Moreover, its blockage may not only compromise MM cell survival and proliferation, but also influence cell adhesion and migration. We sought to elucidate the effects of sorafenib on proliferation, phenotype, specific signalling pathways, actin polymerization and chemotaxis, as well as cytotoxic interactions when combined with other anti-MM agents, such as bortezomib. Methods: L363, U266 and RPMI8226 were cultured with RPMI1640, 10% FCS and 0.2% penicillin/streptomycin. On day 0, cells were treated with increasing concentrations of sorafenib and/or bortezomib. Cell viability and cytotoxicity were assessed on days 3 and 6, in addition to day 1 or 2 in previous analyses. The cytotoxic effect for sorafenib and bortezomib combined was evaluated using Calcusyn Software, whereby a combination index =1, <1 or >1 indicated additive, synergistic and antagonistic effects, respectively. CD138 expression and morphologic changes were evaluated via flow cytometry, immunocytochemistry and confocal microscopy. The effect of sorafenib on ERK1/2 phosphorylation was investigated by western blot. Actin polymerization was studied by flow cytometry after labeling with FITC-phalloidin. Chemokine receptor expression was assessed by flow cytometry and chemotaxis of L363 cells with various chemoattractants was studied using 96-well chemotaxis chambers. Results: Our MM-in vitro model confirmed potent cytotoxicity for sorafenib single use and synergistic effects when combined with bortezomib. With 10 and 100μM sorafenib in L363, we observed increased median PI+ cells (62% and 94% on d3, respectively) compared to the control (median PI+ d0: 11%), with similar increases on d6 (median 81% and 92%, respectively). Combined sorafenib and bortezomib use showed additive effects and synergism at 10μM and 10nM bortezomib (combination index: 0.80). Similar to PI-results, viable cells and CD138 expression by flow cytometry substantially decreased with sorafenib in a dose- and time-dependent manner. Regarding the effects on the MAPK pathway, after incubating L363 cells with 1 and 10μM sorafenib for 6 and 24 hours, a dose-dependent downregulation of ERK1/2 phosphorylation was observed. After 3 days of incubation with increasing concentrations of sorafenib, MM cells were stained with DAPI, Phalloidin-Alexa594 and CD138-FITC and analyzed via confocal microscopy. L363 cells highly expressed CD138 in the absence of sorafenib. Of note, sorafenib not only affected cell proliferation, but also phenotype, morphology, actin metabolism and chemotaxis of MM cells. With sorafenib concentrations as low as 1μM, CD138 was downregulated and impressive morphologic changes with a reduction in F-actin content were observed. We could show CXCL12-stimulated actin polymerization and after treatment with sorafenib with concentrations of 10μM and 100μM its inhibition, as confirmed via flow cytometry after labeling with phalloidin-FITC. L363 cells showed high expression of the chemokine receptors CCR4 and CCR5 and underwent chemotaxis to their common ligand CCL5. Chemotaxis of L363 cells was even more evident with the use of supernatant from M210B4 bone marrow stromal cells. This M210B4-induced chemotaxis also occurred in the presence of the specific CXCR4-inhibitor AMD3100, supporting the involvement of chemokines other than CXCL12 in M210B4-induced MM cell migration. M210B4-triggered chemotaxis was substantially inhibited after 3 days of incubation with increasing concentrations of sorafenib in a dose-dependent manner. Conclusions: To the best of our knowledge this is the first analysis of the effects of sorafenib on phenotype, morphology, actin polymerization and migration of MM cells. Sorafenib induced down-regulation of phospho-ERK appeared responsible for the observed actin depolymerization and reduction in M210B4-triggered chemotaxis. Hence, further analysis of sorafenib and other novel anti-MM agents, both in MM cells and their microenvironment, should enable greater progress in this hematopoietic disease. Disclosures: No relevant conflicts of interest to declare.

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Inhibition Of PI3K Classia Kinases Using GDC0941 Overcomes Protection Of Multiple Myeloma Cells In The Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v122.21.3169.3169 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3169-3169

Author(s):

Hugh Kikuchi ◽

Amofa Eunice ◽

Maeve McEnery ◽

Farzin Farzaneh ◽

Stephen A Schey ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Proliferation ◽

Bone Resorption ◽

Cell Lines ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Pi3k Pathway ◽

Dependent Manner ◽

Dose Dependent

Abstract Despite of newly developed and more efficacious therapies, multiple myeloma (MM) remains incurable as most patient will eventually relapse and become refractory. The bone marrow (BM) microenvironment provides niches that are advantageous for drug resistance. Effective therapies against MM should ideally target the various protective BM niches that promote MM cell survival and relapse. In addition to stromal mesenchymal/myofibroblastic cells, osteoclasts play a key supportive role in MM cell viability. Additionally, 80% of patients develop osteolytic lesions, which is a major cause of morbidity. Increased osteoclast activity is characteristic in these patients and targeting osteoclast function is desirable to improve therapies against MM. Osteoclasts need to form an F-actin containing ring along the cell margin that defines a resorbing compartment where protons and degradative enzymes are secreted for dissolution of bone mineral. Remodelling of F-actin and vesicle secretion are regulated by the class IA PI3K pathway during osteoclastic bone resorption. Additionally, it has recently been shown that inhibition of the class IA PI3K pathway in MM cells with GDC0941 induces apoptosis-mediated killing. We hypothesised that GDC0941 could be used as a therapeutic agent to overcome MM-induced osteoclast activation. GDC0941 inhibited maturation of osteoclasts derived from BM aspirates from MM patients in a dose dependent manner. This correlated with decreased bone resorption of osteoclasts cultured on dentine discs. Exposure of mature osteoclasts to GC0941 resulted in abnormal organisation of larger F-actin rings, suggesting a negative effect on the dynamics of the actin cytoskeleton required for bone resorption. We also found that GDC-0941 can prevent protection of the MM cell lines MM1.S and MM1.R by osteoclasts against killing. GDC-0941 alone blocked MM cell proliferation independently of the presence of BM stromal cells and synergised with other therapeutic agents including Lenalidomide, Pomalidomide, Bortezomid and Dexamethasone. We also found that in the presence of MM cells, Dexamethasone (a drug commonly used alone or in combination with new drugs against MM) induced the proliferation of BM stromal cells and adhesion of MM cells on this protective stroma in a dose dependent manner. Dexamethasone is highly effective at MM cell killing when cells are cultured alone. However, we found that at low doses (below 1 uM) and in the presence of BM stromal cells, Dexamethasone could induce MM cell proliferation. GDC0941 enhanced Dexamethasone killing even in the presence of BM stromal cells by blocking Dexamethasone-induced stromal cell proliferation and adhesion of MM cells on the stroma. Targeting individual the PI3K Class IA isoforms alpha, beta, delta or gamma proved to be a less efficient strategy to enhance Dexamethasone killing. Previous work has shown that efficacy of targeting individual PI3K Class I A isoforms would be low for activation of caspases in MM cells as it would be dependent on relative amounts of isoforms expressed by the MM patient. GDC-0941 also inhibited the proliferation of MM1.R and RPMI8266 MM cell lines, which are less sensitive to treatment to Dexamethasone. Co-culture of MM cells with BM stromal cells induced the secretion of IL-10, IL-6, IL-8, MCP-1 and MIP1-alpha. The dose-dependant increased proliferation of Dexamethasone-treated MM cells in the presence of the BM stroma correlated with the pattern of secretion of IL-10 (a cytokine that can induce B-cell proliferation) and this was blocked by the combination of Dexamethasone with GDC0941. GDC-0941 alone or in combination with Dexamethasone was more efficacious at inducing MM cell apoptosis in the presence of the BM stroma cells vs treatment of MM cells alone. These are very encouraging results as they suggest that GDC-0941 in combination with Dexamethasone would be potentially highly efficacious for targeting MM cells in the BM microenvironment. We are currently performing in vivo data using C57BL/KaLwRij mice injected with 5T33-eGFP MM cells that will be discussed at the meeting. We propose that MM patients with active bony disease may benefit from treatment with GDC0941 alone or in combination with currently used therapeutic drugs against MM. Disclosures: No relevant conflicts of interest to declare.

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Overcoming Innate Resistance to a Beta-Catenin Inhibitor-BC2059- By Manipulating Autophagy in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.5669.5669 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5669-5669

Author(s):

Ioanna Savvidou ◽

Tiffany T. Khong ◽

Stephen K. Horrigan ◽

Andrew Spencer

Keyword(s):

Multiple Myeloma ◽

Myeloma Cell ◽

Autophagic Flux ◽

Beta Catenin ◽

Dependent Manner ◽

Caspase 9 ◽

Innate Resistance ◽

Proliferative Effect ◽

Apoptotic Pathways ◽

Dose Dependent

Abstract Introduction: Chemoresistance is a major challenge in the development of new therapies in multiple myeloma [MM]. Inhibition of autophagy has been shown to restore chemosensitivity in several tumors. We have previously validated a beta-catenin inhibitor (BC2059) which targets the Wnt/beta-catenin signaling pathway. Aim: In the present study we aim to overcome innate resistance to BC2059 by manipulating the autophagic pathway. Methods: Autophagic flux was estimated by measurement of LC3II/LC3I in the absence and presence of hydroxychloroquine [HQ] by Western blot [WB]. Induction of autophagy was measured by the increase of LC3II/LC3I by WB, and concomitant drop of p62 expression by Flow Cytometry [FC]. Combination Indices [CI] were calculated using Calcusyn software. Results: BC2059 induces apoptosis in a dose-dependent manner by induction of both the intrinsic and extrinsic apoptotic pathways, (increase of active -caspase-8, -caspase-9 and -caspase-3 measured by FC and cleaved PARP by WB) in all human myeloma cell lines [HMCL] tested. All HMCL tested have significant autophagic flux at baseline. Chemical inhibition of autophagy has an anti-proliferative effect, decreasing the relative cell numbers from 40% (NCI-H929) to 23% (KMS12BM) at 24hr. In parallel BC2059 is able to induce autophagy in a dose dependent manner. Induction of autophagy is BC2059 specific as treatment with melphalan or bortezomib at relative equal anti-proliferative doses did not increase LC3II/LC3I. Further autophagic inhibition by HQ was synergistic for all HMCL with CI of 0.7-0.3 (CI<1.1 indicates synergy). Interestingly, inhibition of autophagy halved the LD50 of BC2059 in a resistant HMCL (LP1). Other autophagy inhibitors (3-MA, bafilomycin A1 and NH4Cl) were also synergistic with BC2059 in LP1. Conclusion: BC2059 exerts cytotoxicity mainly by induction of apoptosis but also induces cyto-protective autophagy. Autophagy inhibition was able to overcome innate resistance to the drug, ameliorating its cytotoxic effect. This study warrants further investigation. Disclosures Horrigan: betacatpharmaceuticals: Employment.

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Rhein Induces a Necrosis-Apoptosis Switch in Pancreatic Acinar Cells

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2014/404853 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 8

Author(s):

Xianlin Zhao ◽

Juan Li ◽

Shifeng Zhu ◽

Yiling Liu ◽

Jianlei Zhao ◽

...

Keyword(s):

Annexin V ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Maximum Effect ◽

Dependent Manner ◽

Necrotic Cell ◽

Ar42j Cells ◽

Associated Proteins ◽

Apoptosis And Necrosis ◽

Dose Dependent

Objectives. The Chinese herbal medicine Da-Cheng-Qi decoction can regulate a necrosis-apoptosis switch in injured pancreatic acinar cells. This study investigated the effects of rhein, a component of this medicine, on a necrosis-apoptosis switch in pancreatic rat AR42J cells.Methods. Cerulein-treated AR42J cells were used. After pretreatment with 479, 119.8, or 29.9 μg/L rhein, cells were cocultured with rhein and cerulein (10−8 M) for 4, 8, or 16 h. Apoptosis and necrosis were examined using annexin V and propidium iodide costaining. Mitochondria-dependent apoptosis-associated proteins were examined using enzyme-linked immunosorbent assays and western blotting.Results. Few cells died in untreated samples. The number was significantly higher in 16-h-cerulein-treated samples and treatment with 479 μg/L rhein most effectively increased the apoptotic-to-necrotic cell ratio (P<0.05). In cerulein-treated cells, rhein increased the concentrations of p53, cytochrome C, and caspase-3, and increased the Bax/Bcl-2 ratio in a time- and dose-dependent manner, with the maximum effect in cells treated with 479 μg/L rhein for 16 h (P<0.05).Conclusions. Rhein induces the necrosis-apoptosis switch in injured pancreatic acinar cells in a time- and dose-dependent manner. Mitochondria-dependent apoptosis signaling pathways might play an important role in this effect.

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An Immune Based, Anti-CD138 Targeting Antibody for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-119112 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5617-5617 ◽

Cited By ~ 3

Author(s):

Tengteng Yu ◽

Lijie Xing ◽

Liang Lin ◽

Jiye Liu ◽

Kenneth Wen ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Myeloma Cell ◽

Dependent Manner ◽

Cellular Cytotoxicity ◽

Adcc Activity ◽

Effector Cells ◽

Equity Ownership ◽

Dose Dependent

Abstract CD138 (Syndecan-1), a member of integral membrane family of heparan sulfate proteoglycans (HSPGS), is highly expressed on differentiated plasma cells (PC) and is both a primary diagnostic biomarker of multiple myeloma (MM) as well as an indicator of poor clinical prognosis. This surface antigen is an attractive candidate for targeted immunotherapy for MM, given its constitutive expression during disease progression, including smoldering myeloma, a relatively early asymptomatic phase of disease that is potentially amenable to early treatment. We here investigated the targeted use of chimeric anti-CD138 monoclonal antibody (mAb) 1610 and confirm its in vitro anti-tumor potency based on an immune directed cellular cytotoxicity against a diverse panel of CD138 positive MM cell lines, both resistant or sensitive to conventional and current MM therapies and varying levels of CD138 expression as measured by cell immunostaining and quantitative RT-PCR. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using a calcein-AM based release assay in the presence of human natural killer (NK) effector cells purified from four different healthy donors. MAb 1610 lysed CD138-expressing MM cell lines in a dose dependent manner. This ADCC activity was mAb 1610 specific (in comparison to isotype control), CD138 target dependent, and mediated in the presence of human NK effector cells (co-cultured at an effector:target cell ratio of 20:1). MAb 1610 dependent-cytotoxicity was observed at concentrations as low as 0.01 µg/ml with maximal lysis occurring at approximately 1 µg/ml and extrapolated sub-nanomolar ED50 potencies (Table 1) based on these data. All MM cell lines were subject to mAb 1610-mediated lysis, albeit with slightly different sensitivities that modestly correlated with their relative CD138 cell surface expression levels. This anti CD138 mAb-dependent cellular toxicity included MM1SR and H929R cell lines, both of which are resistant to lenalidomide. MAb 1610 induced specific cell lysis of JJN3 cells, but not of CD138 knock out JJN3 cells or CD138-negative B lymphocytes, further confirming that mAb 1610 specifically induced ADCC against-CD138 expressing MM cells in a target specific manner. Using an orthogonal cytometric based assay, the ability of mAb 1610, in a dose-dependent manner, to activate NK cells was also shown in the presence of CD138 target cells, as evidenced by increased expression of CD107 (a marker for NK cell degranulation) and cytokine production in NK cells. Importantly, the CD138 targeting cytotoxic activities of mAb 1610 translationally extend to MM cells autologously derived directly from MM patients with newly diagnosed and relapsed/refractory diseases. The concomitant use of autologously derived effector cells from these patients to mediate antibody dependent myeloma cell killing further suggests the relevance of anti-CD138 directed immune-based therapeutic strategy in humans. In further replication of human disease, we also co-cultured MM1.S or MM1.R cells with human bone marrow stromal cells (BMSCs) which support myeloma cell growth by promoting an immunosuppressive microenvironment within the BM. Importantly, mAb 1610-dependent cytotoxicity against MM1.S or MM1.R cells was not attenuated by the co-presence of BMSCs. Similarly, IL-6 (10 ng/ml) did not significantly affect mAb 1610-induced ADCC activity, indicating a mechanism of action that can overcome growth promotion, immune suppression, and drug resistance conferred by the tumor promoting BM microenvironment. Taken together, these in vitro studies further demonstrate as a proof-of-concept the use of an antibody CD138 targeting strategy mediated through an immune based mechanism of myeloma plasma cell killing. Based on these results, optimization and further biological characterization of chimeric mAb 1610 in advance of pre-clinical studies is anticipated. Disclosures Myette: Visterra Inc.: Employment. Chaganty:Visterra Inc.: Employment. Adari:Visterra Inc.: Employment. Tissire:Visterra Inc.: Employment. Deotale:Visterra Inc.: Employment. Shriver:Visterra Inc.: Employment. Munshi:OncoPep: Other: Board of director. Anderson:Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy; Bristol Myers Squibb: Consultancy.

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Impacts of a Proliferation-Inducing Ligand on Current Therapeutic Monoclonal Antibody-Induced Cytotoxicity Against Human Multiple Myeloma Cells

Blood ◽

10.1182/blood-2019-130927 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3105-3105 ◽

Cited By ~ 1

Author(s):

Liang Lin ◽

Shih-Feng Cho ◽

Kenneth Wen ◽

Tengteng Yu ◽

Phillip A Hsieh ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Cell Membrane ◽

Nk Cell ◽

Cell Lysis ◽

Advisory Board ◽

Dependent Manner ◽

Effector Cells ◽

Human Multiple Myeloma ◽

Dose Dependent

A proliferation inducing ligand (APRIL) is a natural ligand for B cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI), two receptors overexpressed in human multiple myeloma (MM) patient cells. Specifically, BCMA is highly expressed in plasma cells of all MM patients and BCMA-based immunotherapies has recently shown impressive response rates in patients with relapsed and refractory diseases. APRIL, mainly secreted by myeloma-supporting bone marrow (BM) accessory cells, i.e., macrophages, osteoclasts (OC), promotes MM cell progression in vitro and in vivo. It further induces survival and function of regulatory T cells (Treg) via TACI, but not BCMA, to support an immunosuppressive MM BM microenvironment (Leukemia. 2019;33:426). Here, we study effects of APRIL in current immunotherapies in MM and determine whether APRIL influences antibody-dependent cellular cytotoxicity (ADCC) induced by therapeutic anti-BCMA (J6M0) or anti-CD38 (daratumumab) mAbs via FcR-expressing immune effector cell-dependent mechanisms. Using anti-human IgG1 to detect J6M0 binding to the cell membrane BCMA, we first showed that APRIL, in a dose-dependent manner (31-500 ng/ml), competed with J6M0 for binding to BCMA. Such effects were inhibited by the blocking anti-APRIL monoclonal antibody (mAb) (Apry-1-1), as confirmed by flow cytometry and confocal microscopy. APRIL still inhibited J6M0 binding to BCMA at 4°C, arguing against that APRIL induces shedding of BCMA receptor. Using PE labeled anti-FLAG to detect APRIL-FLAG bindings to MM cell surface BCMA, J6M0 (0.25-4 µg/ml) did not alter APRIL binding to BCMA following 2h or 1d incubation. High concentrations of J6M0 (>10 µg/ml) only blocked ~50% of APRIL (0.2 µg/ml)-induced NFκB activity as determined by specific DNA binding assays, indicating that APRIL-induced signaling cascade via BCMA or TACI in MM cells is not completely blocked by J6M0. In parallel, data analysis using mRNA-seq identified 594 or 355 differentially expressed genes (Log2-Fold-change > 1.5 and adjusted p < 0.05) in APRIL- and BCMA-overexpressed RPMI8226 MM cell transfectants, respectively, when compared with control parental cells. KEGG and Reactome pathway enrichment analysis further defined that these differentially expressed genes are enriched in cell adhesion, migration, chemokine signaling pathways, and JAK/STAT signaling pathways, in addition to proliferation and survival in MM cells. We next asked whether overnight treatment with APRIL in MM cell lines decreased their baseline lysis by FcR-expressing effector cells, i.e., NK, monocytes. In a dose-dependent manner, APRIL (10-200 ng/ml) downregulated baseline MM cell lysis mediated by these effector cells. Importantly, in a similar fashion, ADCC was decreased against all APRIL-treated vs control MM cell lines induced by J6M0 or daratumumab. Conversely, blocking anti-APRIL mAbs reverted APRIL-suppressed cytotoxicity against MM cells induced by J6M0 or daratuzumab. These results were validated by decreased J6M0-induced NK cell degranulation following co-incubation with APRIL-treated vs control MM cells. In contrast, anti-APRIL neutralizing mAbs specifically blocked APRIL-inhibited NK cell membrane CD107a expression. Furthermore, co-cultures with MM-supporting OCs or macrophages decreased ADCC against MM cells by NK cells; conversely the neutralizing anti-APRIL mAb significantly blocked APRIL-reduced MM cell lysis by J6M0- or Daratumumab. Finally, APRIL reduced J6M0-induced patient MM cell lysis when freshly isolated BM mononuclear cells from MM patients (n=10) were incubated with NK cells from the same individual. Anti-APRIL mAbs still blocked APRIL blockade in J6M0-induced autologous patient MM cell lysis. Taken together, our data further indicate that therapies directed at the APRIL/BCMA and APRIL/TACI axes may simultaneously target MM cells and counteract APRIL-reduced MM cell lysis induced by therapeutic mAbs targeting MM cells. These results thus support combination strategies of blocking APRIL mAbs with BCMA- or CD38-directed immunotherapies to further overcome MM cell-induced immunosuppressive BM microenvironment, thereby enhance Disclosures Munshi: Abbvie: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Adaptive: Consultancy; Celgene: Consultancy. Anderson:Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder .

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