scholarly journals The Role of CD38 in Multiple Myeloma Cell Biology

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1580-1580
Author(s):  
Yawara Kawano ◽  
Saki Kushima ◽  
Hiroyuki Hata ◽  
Masao Matsuoka
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Cell Biology ◽  
Proteome Analysis ◽  
Cd38 Expression ◽  
Mammalian Tissues ◽  
Nadh Ratio

Abstract Introduction. Anti-CD38 monoclonal antibodies, such as daratumumab and isatuximab, which exerts therapeutic effect against multiple myeloma (MM) cells through direct cell damage, antibody dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), has shown its high efficacy in clinical practice. However, the role of CD38 in MM cell biology is still unclear. CD38 is known as a major nicotinamide adenine dinucleotide (NAD +) glycohydrase (NADase) in mammalian tissues, which regulate cellular levels of NAD +. In the present study, we compared metabolic and proteomic profile between CD38 positive and negative MM cell lines to analyze the biological significance of CD38 in MM cells. Additionally, we performed CD38 enzyme activity inhibition on MM cells using 78c, a NADase enzyme inhibitor of CD38, in order to study the role of CD38 NADase activity in MM cell survival. Materials and methods. MM cell lines harboring CD38 positive and negative fractions (KMS-12BM, KMS-11) were sorted according to CD38 expression. Intracellular NAD+ and NADH concentrations between CD38 positive and negative cells were analyzed using NAD/NADH Assay kit. CD38 positive and negative MM cells were subjected to metabolome and proteome analysis using Shimadzu TQ8050 GC-MS/MS and TripleTOF 5600 respectively. Metabolites and proteins significantly enriched in CD38 negative MM cells were analyzed using MetaboAnalyst and Metascape. Cell cycle status between CD38 positive and negative cells were determined by flow cytometry after staining by BRDU and 7AAD. CD38 positive MM cell lines (NCI-H929 and KMS-12PE) and patient derived bone marrow cells were treated with 78c, a CD38 NADase inhibitor, in vitro. MM cell viability were determined by flow cytometry post Annexin V and PI staining. Differences in metabolites between 78c treated and control MM cell lines were also analyzed using Shimadzu TQ8050 GC-MS/MS. Results. Higher NAD+ and NAD+/NADH ratio was observed in CD38 negative fraction of MM cell lines compared to their CD38 positive counterparts, demonstrating that cell surface CD38 expression influences intracellular NAD+ concentration. Both metabolome and proteome analysis revealed that CD38 negative cells tend to have higher glycolytic activity compared to CD38 positive cells. Significant suppression of cell cycle, accompanying G0/G1 phase arrest, was observed in CD38 negative MM cells, indicating that metabolic shift in CD38 negative MM cells may lead to change in cell proliferation. Marked increase of NAD+/NADH ratio was observed in 78c treated MM cell lines compared to control, proving that CD38 NADase inhibiton indeed affects intracellular NAD+ concentration in MM cells. 78c was capable of inducing cell death in MM cell lines and patient derived MM cells, accompanying cell cycle arrest. Metabolites significantly upregulated in 78c treated MM cells compared to control were associated with glycolysis, demonstrating that CD38 NADase activity has a significant effect on MM cell metabolism. Conclusions. CD38 is the major NADase in mammalian tissues, involved in catabolism of NAD +. Although CD38 is highly expressed in normal plasma cells and MM cells, its role in MM cell biology has not been studied in detail. By comparing CD38 positive and negative cells and using CD38 NADase inhibitor, we showed for the first time that CD38 on MM cells decrease intracellular NAD+, reduces intracellular glycolysis and as a result, has an influence on cell cycle. The present study sheds light on the significance of CD38 enzyme activity in MM cell biology and may also contribute to understanding the mechanism of resistance to CD38 targeted therapy. Disclosures Kawano: Janssen Pharmaceuticals: Honoraria; Ono pharmaceutical: Honoraria; Sanofi: Honoraria; Bristol Myers Squibb: Honoraria; Takeda Pharmaceuticals: Honoraria.

scholarly journals DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5618-5618 ◽  
Author(s):  
Priya Choudhry ◽  
Margarette C. Mariano ◽  
Arun P Wiita
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Ex Vivo ◽  
Single Agent ◽  
Cd38 Expression

Abstract Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy. Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega). Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here. Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

scholarly journals Targeting Nicotinamide Adenine Dinucleotide (NAD) Glycohydrase Activity of CD38 Exerts Anti-Myeloma Effect Accompanying Intracellular NAD Elevation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1810-1810
Author(s):  
Saki Kushima ◽  
Takayuki Sasano ◽  
Masao Matsuoka ◽  
Hiroyuki Hata ◽  
Yawara Kawano
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cell Lines ◽  
Nicotinamide Adenine Dinucleotide ◽  
Research Funding ◽  
Therapeutic Strategy ◽  
Cd38 Expression ◽  
Low Concentrations ◽  
Mammalian Tissues

Introduction. The development of novel agents has improved the outcomes of multiple myeloma (MM) patients. Especially, daratumumab, an anti-CD38 monoclonal antibody which exerts therapeutic effect against MM cells through direct cell damage, antibody dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), has shown its high efficacy in clinical practice. CD38 is a transmembrane glycoprotein highly expressed in plasma cells. CD38 is also a major nicotinamide adenine dinucleotide (NAD) glycohydrase in mammalian tissues, which regulate cellular levels of NAD. However, the role of CD38 as a NAD glycohydrase (NADase) in survival of MM cells is not well understood. In the present study, we conducted CD38 enzyme activity inhibition on MM cells using a small molecule compound 78c, a specific inhibitor for NADase enzymatic activity of CD38, in order to study the role of CD38 NADase activity in MM cell survival and to examine whether CD38 enzyme inhibition could be a new therapeutic strategy of MM. Materials and methods. MM cell lines (NCI-H929, KMS-12BM, KMS-12PE, U266) were treated with CD38 NADase inhibitor, 78c, in vitro. Viability of MM cell lines and patient-derived MM cells were analyzed by flow cytometry after 7AAD staining. MM cell lines possessing CD38 positive and negative fraction were sorted according to the CD38 expression using CD38 Micro-Beads. CD38 low MM cell lines were treated with All-trans retinoic acid(ATRA)to increase surface CD38 expression. Intracellular NAD and NADH concentrations in MM cells were analyzed using NAD / NADH assay kit. Detection of apoptosis in MM cell lines were examined by Annexin V and PI staining followed by flow cytometry analysis. Caspase inhibitor, Z-VAD-FMK, was used in combination with 78c to study the mechanism of 78c induced MM cell death. Results. 78c induced cell death in MM cell lines at low concentrations (IC50 10-20 μM). Addition of 78c to patient derived bone marrow cells showed cytotoxicity to MM cells, while toxicity to non-MM cells were limited. CD38 positive fraction of MM cell lines had better sensitivity to 78c compared to CD38 negative fraction. CD38 induction by ATRA in CD38 low MM cell lines showed increased sensitivity to 78c. These results proved that 78c efficacy correlates with surface CD38 expression. Comparison of intracellular NAD and NADH concentrations between CD38 positive and negative fractions of MM cell lines demonstrated a significant increase of NAD in the CD38 negative fraction compared to their positive counterparts, indicating that CD38 is indeed controlling the intracellular NAD concentration. Marked increase of NAD / NADH ratio was observed in 78c treated MM cell lines compared to control, proving that CD38 NADase inhibition affects intracellular NAD concentration in MM cells (Fig. 1). 78c treatment of MM cell lines significantly reduced the number of viable cells in the Annexin- / PI- region, however, addition of Z-VAD-FMK did not lead to recovery of viable cell numbers, indicating non-apototic cell death induction by CD38 NADase inhibition. Conclusions. CD38 is the major NADase in mammalian tissues, and involved in catabolism of NAD. CD38 NADase inhibitor, 78c, inhibited the growth of MM cells at low concentrations. 78c induced cell death was found to be highly specific to MM cells and its cytotoxic effect was associated with surface CD38 expression of MM cells. Increased amount of NAD in MM cells by 78c treatment suggests that NAD elevation is associated with MM cell death induced by CD38 NADase inhibition. Since, daratumumab has limited effect against CD38 NADase activity, modulation of intracellular NAD levels by CD38 NADase inhibition could provide a novel therapeutic strategy for MM (Fig. 2). Disclosures Matsuoka: Kyowa Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb Corp.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.

Aptamer TY04 Inhibits Multiple Myeloma Cell Growth Via Cell Cycle Arrest

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5390-5390
Author(s):  
Jing Liu ◽  
Hong-Juan Dai ◽  
Bian-Ying Ma ◽  
Jian-Hui Song ◽  
Hui-yong Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Growth ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
M Phase ◽  
Target Molecules

Abstract Multiple myeloma (MM), also known as plasma cell myeloma, is characterized by accumulation of clonal plasma cells in the bone marrow and overproduction of monoclonal immunoglobulin (Ig) in the blood or urine. MM accounts for approximately 10% of all hematologic malignancies. Despite recent advances in the understanding and treatment of this disease, MM remains an incurable disease in the vast majority. With conventional chemotherapy, the 5-year median survival rate for MM patients is approximately 25%. Aptamers are single-stranded RNA or DNA sequences that bind to target molecules with high affinity and specificity. Compared with antibodies, aptamers have unique advantages including easy chemical synthesis and modification, low toxicity, lack of immunogenicity, and rapid tissue penetration, Based on these advantages, aptamers show great potential for therapeutic application. The aptamer TY04 is a single-stranded DNA (ssDNA) generated by a method named cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX), We found TY04 strongly inhibited the growth of multiple myeloma cell lines including MM1.S, NCI-H929, KM3 and OPM2,The concentration of TY04 to inhibit 50% cell growth (IC50) on MM1.S was 3.89 μM. In contrast, TY04 had no effect on the growth of non-tumor cell lines — immortal B lymphoblastoid cell lines. Next, we used MM1.S cell line as the model to study the mechanism of TY04 anti- multiple myeloma. Flow cytometry analysis showed that TY04 with the sequence specifically bind to MM1.S cells when compared with unselected ssDNA library control. To investigate whether the target molecules of TY04 are membrane proteins on cell surface, MM1.S cells were treated with trypsin and proteinase k for 2 or 10 minutes before incubation with TY04. The result revealed that TY04 lost partly recognition ability on treated cells, indicating that the target molecules were most likely membrane proteins. Furthermore, we evaluated the cell cycle distribution of MM1.S after TY04 treatment. We found that TY04 significantly caused cell-cycle arrest in G2/M phase. The percentage of G2/M phase cells increased from 30.1±1.56 to 53.2±6.36. To identify the underlying molecular mechanism, G2/M-related proteins were assayed by flow cytometry. Following TY04 treatment, a concomitant inhibition of ERK1/2, cyclin B, CDK1 and γ-tubulin expression occurred. Meanwhile, human cell cycle PCR array was used to analyze the expression of 84 genes key to cell cycle regulation in TY04-treated MM1.S cells. Our results indicated that aptamer TY04 decreased the genes expression of CCNB1, CCNB2, BIRC5, BRCA1 and CCNH, which were involved in the progress of G2/M phase. All these results are significant in that they provide a framework for further exploring the use of TY04 as a novel anti-multiple myeloma agent. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Tris DBA Palladium Overcomes Hypoxia Mediated Drug Resistance in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2978-2978
Author(s):  
Pilar De La Puente ◽  
Barbara Muz ◽  
Feda Azab ◽  
Micah John Luderer ◽  
Jack L. Arbiser ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Drug Resistance ◽  
Cell Lines ◽  
Research Funding ◽  
Caspase 3 ◽  
Cellular Processes ◽  
Hypoxic Conditions

Abstract Introduction: Despite recent progress in novel and targeted therapies, multiple myeloma (MM) remains a therapeutically challenging incurable disease. The regulation of important cellular processes and its link to cancer presented Src as an attractive target for MM. Src is a non-receptor protein tyrosine kinase which regulates multiple fundamental cellular processes including cell growth, migration, survival and differentiation. Activated Src in cancer lead to studies with Src as a target for anti-cancer drugs, and numerous Src inhibitors have become available to test the importance of Src in tumor initiation and progression. In MM, it has been described that in cell lines and MM patient-derived tumors, c-Src is constitutively activated, which plays an important role in drug resistance mechanisms. Tris dibenzylideneacetone dipalladium (Tris DBA), a small-molecule palladium complex, was shown to reduce Src/NMT-1 complex in melanoma cells, as well as inhibit downstream signaling including mitogen-activated protein kinase (MAPK kinase) and phosphoinositol-3-kinase (PI3K). We suggest a novel strategy to improve the treatment of MM and overcome the drug resistance for the current therapeutic agents by specific inhibition of Src in MM cells by an organopalladium compound, Tris DBA. Methods: Tris DBA was prepared by Dr. Arbiser. MM cell lines (MM.1S, MM.1R, H929, RPMI-8826, and OPM2) and PBMCs were cultured with Tris DBA (0-10 µM) for 24h. MM cells were analyzed for cell proliferation by MTT assay; cell cycle by DNA staining with PI and analyzed by flow cytometry; apoptosis was analyzed by Annexin V/PI staining and analyzed by flow cytometry; and cell signaling associated with proliferation, cell cycle, and apoptosis was analyzed by western blotting. In addition, cell proliferation assay of Tris DBA with or without combination of proteasome inhibitors (PIs) bortezomib or carfilzomib for 24h was analyzed on the proliferation of MM cells in normoxic or hypoxic conditions. Moreover, we tested the effect of combination treatment on cell cycle and apoptosis signaling under normoxic conditions. We then evaluated the effect of Tris DBA on HIF1α expression, migration and drug resistance under normoxic or hypoxic conditions. Results: The Src inhibitor Tris DBA reduced the proliferation of MM cell lines with an IC50 of about 1.5 - 3 µM after 24h treatment as a single agent, while none of the normal PBMC controls showed effect on their proliferation in the same dose range. These results were consistent with the decreased expression of proliferation signaling proteins from MAPK pathways (pERK), as well as PI3K (pS6R). Src inhibition led to the induction of a sub-G1 peak, which indicated accumulating apoptotic cells shown by DNA staining with PI. Apoptosis was then analyzed by Annexin/PI and confirmed by cleavage of caspase-3 and PARP. We found that Tris DBA synergized with bortezomib and carfilzomib by inhibiting proliferation of MM cells and reducing cell cycle protein signaling more than either of the drugs alone. Moreover, the Tris DBA/Bortezomib or Tris DBA/Carfilzomib combination therapies significantly increased apoptosis by caspase-3 cleavage more than treatment with either proteasome inhibitor individually. Tris DBA inhibited HIF1α expression in both normoxic and hypoxic conditions. HIF1α is an important target for hypoxia-driven drug resistance. Our studies confirmed hypoxia promoted faster chemotaxis of MM cells towards the chemo-attractants found in stromal cell conditioned media, and that Tris DBA treatment could overcome this hypoxia-induced effect. In addition, the development of hypoxia-induced drug resistance to individual bortezomib or carfilzomib treatment was overcome with combination treatment of Tris DBA under hypoxic conditions. Conclusions: Tris DBA reduces proliferation and induces G1 arrest and apoptosis in MM cells. Tris DBA synergized with PIs reducing proliferation and cell cycle signaling, as well as increasing apoptosis more than each drug alone. Tris DBA overcame hypoxia-induced effects such as enhanced chemotaxis or drug resistance to PIs by inhibition of HIF1α expression. Moreover, we found that Tris DBA is an effective anti-myeloma agent alone or in combination with other targeted drugs and that it reverses hypoxia-induced drug resistance in myeloma. These results suggest the use of Tris DBA as a new therapeutic agent in relapsed refractory myeloma. Disclosures Arbiser: ABBY Therapeutics: Other: Jack L Arbiser is listed as inventor on a US Patent for imipramine blue. He is cofounder of ABBY Therapeutics, which has licensed imipramine blue from Emory University.. Azab:Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding.

scholarly journals High Glycine Promotes Proliferation and Progression Though Increase of Glutathione Synthesis in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1791-1791
Author(s):  
Jiliang Xia ◽  
Jingyu Zhang ◽  
Bin Meng ◽  
Xuan Wu ◽  
Qian Lei ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Colony Formation ◽  
Clinical Characteristics ◽  
Metabolic Flux ◽  
Conflicts Of Interest

Background: Metabolites in tumor microenvironment have been confirmed to contribute to cancer progression. Our previous untargeted metabolomics study has indicated that glycine was significantly increased in bone marrow and peripheral blood derived from Multiple Myeloma(MM) patients compared with health donors(HD). However, the role of glycine in MM progression and its underling mechanisms remain unclear. Materials and Methods: Liquid chromatography-mass spectrometry (LC-MS) was used to detect the concentration of glycine in peripheral blood derived from (25) MM patients and (21) HD. Metabolic flux experiment was performed to explore the distribution of exogenous glycine in MM cell lines ARP1 and 5TGM1. Soft agar colony formation and cell cycle assay were performed to detect MM cells proliferation. 5TGM1 MM mouse models were prepared to examined the effect of glycine on MM in vivo. The unpaired t test was used to evaluate the difference between two different groups. Two-sided Fisher's exact tests were used to assess the associations between glycine abundance and clinical characteristics in MM patients, with a confidence coefficient (confidence interval, CI) of 95%. Results: Targeted metabolic assay of glycine in peripheral blood confirmed that glycine was significantly higher in MM patients than HD(HD vs. MM patients, 14000 vs. 15200, p=0.047). To explore the role of high glycine in MM progression, the associations between glycine abundance and clinical characteristics were investigated. We found that MM patients with high glycine had significantly higher plasma cells percentage(High glycine vs. Low glycine, 11.00% vs. 27.95, p=0.039) and lower hemoglobin concentration(High glycine vs. Low glycine, 96g/l vs. 77g/l, p=0.016). Moreover, high glycine was found to associate with bone damage(p=0.031). Additionally, colony formation and cell cycle assay results showed Glycine-free RPMI 1640 media inhibited MM cells proliferation. Furthermore, 5TMG1 MM mouse fed with glycine-deficiency fodder had slower progression as compared with 5TMG1 MM mouse fed with normal fodder(p=0.0007). These data suggested that exogenous glycine contributes to MM progression. To characterize how exogenous glycine is metabolized in MM cells, MM cell lines ARP1 and 5TGM1 were cultured in the presence of uniformly labeled 13C-glycine for 2, 4, and 6 hours, then the concentration of glycine metabolism related metabolites in conditional media and MM cells were tested by using LC-MS. As a result, 13C-glycine derived GSH was observed in ARP1 as well as 5TGM1, accounting for 37.2% and 52.7% of total GSH after 6 hours of culture, respectively, alternatively, the levels of 13C-GSH in both cell lines were up-regulated with the extension of culture time, indicating that exogenous glycine was involved in GSH synthesis in MM cells. Furthermore, addition of GSH(10 uM) to glycine-free RPMI 1640 media recover the proliferation ability of ARP1 and 5TGM1. Interestingly, betaine, a competitive similar of glycine, was found to suppress MM cell proliferation, and addition of GSH partially counteracted the effect of betaine on MM cells. Conclusion: These findings thus indicate that glycine promotes MM proliferation in vivo and in vitro, and GSH synthesis is the main metabolic pathway contributing to proliferation. Pharmacological blockage of glycine uptake and utilization shows therapeutic potential in MM treatment. Disclosures No relevant conflicts of interest to declare.

Junctional Adhesion Molecule-A/ F11 Receptor (JAM-A/ F11R) Expression in Multiple Myeloma (MM): a Candidate Biomarker of Aggressive Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2830-2830
Author(s):  
Bani M Azari ◽  
Marc J. Braunstein ◽  
H. Uwe Kluppelberg ◽  
Sadeaqua S Scott ◽  
Eric LP Smith ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Tumor Cells ◽  
Cell Lines ◽  
Adhesion Molecule ◽  
Mrna Levels

Abstract Abstract 2830 Poster Board II-806 Background: Multiple myeloma (MM) is an incurable disease of clonal plasma cells that accumulate in the bone marrow (BM), causing monoclonal IG production, bone marrow failure, osteolytic lesions and kidney disease. Although initially treatable, MM ultimately becomes refractory to treatment, and is invariably fatal, when tumor cells that harbor genetic mutations expand without regulation. Therefore novel treatment targets need to be identified. A key mechanism in MM pathogenesis is regulation of tumor growth by the bone marrow (BM) microenvironment, particularly by bone marrow neo-vascularization and adhesion of tumor cells to the marrow stroma. Aberrantly expressed genes that regulate angiogenesis by MM cells enhance MM progression and constitute targets in its treatment. JAM-A/F11R is an endothelial cell (EC) adhesion molecule of the immunoglobulin superfamily which is a multifunctional cell membrane protein that mediates intracellular signaling events that alter EC migration and paracellular permeability. For example, in breast cancer, attenuation of JAM-A increases tumor invasion and metastasis through a decrease in tumor adhesion (Ulas Naik Cell Adh Migr. 2008 Oct;2(4):249-51.). In this study we explored the JAM-A/F11R expression in MM tumor cells and in patients to determine the potential role of this molecule in the pathogenesis and progression of MM. Methods: The MM cell lines examined were RPMI-8266, U266, and NCI-H929. Human umbilical vein endothelial cells (HUVECs) served as controls. Informed consent was obtained from patients and control subjects. Primary BM tumor cells were enriched to > 95% CD138+ cells by positive selection using anti-CD138 MACS MicroBeads. The CD138-negative fraction was used for outgrowth of confluent EPCs (> 98% vWF/CD133/KDR+). JAM-A mRNA expression was assessed using an microarray gene expression profile, JAM-A probe based real-time PCR, and JAM-A levels in each sample were measure using a standard curve and normalized to GADPH. JAM-A protein levels in MM cell lines and primary tumor cells were measured by flow cytometry and immunofluorescence. For serum studies, peripheral blood was obtained from 25 newly diagnosed MM patients and 8 healthy, age- and sex-matched controls, and JAM-A levels were measured using an ELISA. Statistical analysis was performed using Student's t-test, two-tailed, with P ' .05 considered significant. Results: JAM-A mRNA levels were significantly increased in MM cell lines RPMI-8266, U266, and NCI-H929 compared to HUVECs (U266, P = 3×10-5; RPM1-8266, P = 1×10-6; NC1-H929, P= 5×10-4). The JAM-A mRNA levels were significantly greater in RPMI-8226; P < .04 compared to TNFα-activated HUVECs for 24 hours which is a proangiogenic switch for HUVEC gene expression. The elevated mRNA expression of the JAM-A in MM cell lines was confirmed by immunofluorescence and flow cytometry which showed the presence of both membrane and cytoplasmic JAM-A protein. Microarray analysis of gene expression profiles from 20 patients' corresponding tumor cells and microenvironmental EPCs showed that JAM-A had a higher level of expression in tumor cells versus MM EPC by 12.62 fold, (P=.0000642). Furthermore, JAM-A had a higher level of expression in MM EPC versus normal control EPC by 2.41 fold, (P=.00113) reflecting a complex regulatory role of F11 signaling in MM, similar to breast cancer (Naik, U. et al 2008). JAM-A was also found to be 12.6 fold greater in tumor cells compared to EPCS (P=.0000642). In addition, circulating levels of soluble JAM-A were found to be significantly greater in the serum of MM patients compared to controls (P < .005), with an average 2-fold increase. Serum levels of JAM-A in MM patients also decreased 71% with treatment n=5, P<.05. Conclusion: We show for the first time that JAM-A expression is highly elevated in MM tumor cells and its levels respond to treatment. In addition, MM patients have higher circulating JAM-A levels compared to healthy individuals and circulating JAM-A levels were reduced following treatment, suggesting that JAM-A may serve as a novel biomarker in MM. Current studies in the lab are aimed at correlating these levels with clinical parameters to determine whether JAM-A levels reflect disease severity and response to treatment. Results of these analyses, as well as results of ongoing experiments using JAM-A siRNA and antibody-inhibition approaches to target JAM-A in myeloma tumor and ECs will be presented. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Role of PHF19 As a Promoter of Tumorigenicity and Therapeutic Target in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 508-508
Author(s):  
Carolina D. Schinke ◽  
Pingping Qu ◽  
Shmuel Yaccoby ◽  
Valeriy V Lyzogubov ◽  
Veronica MacLeod ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Candidate Gene ◽  
Cell Lines ◽  
Crucial Role ◽  
Plasma Cells ◽  
Total Therapy

Introduction - Multiple Myeloma (MM) is a hematologic malignancy characterized by clonal growth of differentiated plasma cells (PCs). Despite improvement in MM therapy, the disease remains mostly incurable and is characterized by recurrent relapses with development of resistant clones that eventually lead to patient death. The pathways that lead to resistant and aggressive MM are not fully understood highlighting the need to improve our understanding of MM biology to identify potential new pathways and therapeutical targets. PHD Finger Protein 19 (PHF19) is a regulator of Polycomb Repressive Complex 2 (PRC2), the sole methyltransferase complex capable of catalyzing H3K27me3 to induce and enforce gene repression. PRC2 employs enhancer of zeste homolog 1 and 2 (EZH1/EZH2) as enzymatic subunits to hypermethylate H3K27. While overexpression and gain of function mutations of EZH1/2 have been observed in many cancers the role of this particular pathway in MM remains poorly understood. In the present study, we report on PHF19 as a new candidate gene to play a potential crucial role in MM oncogenesis. Methods- Gene expression profiling (GEP; Affymetrix U133 Plus 2.0) was performed on 739 MM patients (from total therapy trials [TT] 3-5; low risk MM n=636, high risk MM n=103), 42 patients with monoclonal gammopathy of undetermined significance (MGUS), 73 smoldering MM patients, 42 patients with primary plasma cell leukemia and 34 healthy donors. Myeloma risk was determined by the GEP 70 signature as previously defined. To test the implications of functional PHF19 knock down (KD) we used TRIPZ inducible PHF19 shRNA vs. scrambled control (Dharmacon) in two MM cell lines (JJN3 and ARP1). Real time PCR as well as western blotting was used to confirm PHF19 KD as well as to elucidate the effect on H3K27me3 (Cell Signaling). Functional in vitro studies included proliferation (Promega), clonogenic assays (StemCell), cell cycle and apoptosis assays (both Invitrogen). In vivo studies were performed using SCID mice that were subjected to tail vain injection with PHF19 KD JJN3 cells (n=10) or scrambled shRNA control (n=10). Weekly ELISA (Bethyl) and in vivo imaging (Xenogen) were performed and survival was recorded. Results- GEP of the previously mentioned patient populations and healthy controls identified PHF19 (chr9q33.2) as a candidate gene that was consistently dysregulated in MM patients. Mean expression levels at different MM stages correlated with disease aggressiveness (ANOVA, p&lt;0.0001), Figure 1. High expression of PHF19 (log2&gt;10.46) at diagnosis correlated significantly with adverse clinical parameters, including ISS III, anemia and elevated LDH, as well as worse overall survival (5 yr OS = 29% for patients with high PHF19 expression vs 77% for patients with low PHF19 expression [log2&lt;10.46], p&lt; 0.0001). These results led us to test the implications of functional PHF19 KD using TRIPZ inducible PHF19 shRNA vs. scrambled control in the JJN3 and ARP1 MM cell lines. PHF19 KD led to a drastic reduction of H3K27me3 thereby resulting in significantly reduced proliferation via cell cycle arrest, while apoptosis was not substantially altered. Clonogenic assays showed a significant reduction in colony numbers and size of MM cells with PHF19 KD compared to the control (&gt;75% reduction in both cell lines, p&lt;0.05). Xenograft studies showed consistently less tumor burden in the mice injected with PHF19 KD cells compared to scrambled control, evident through ELISA testing for IgG Kappa (Median =180 mg/ml for scrambled control vs 80 mg/ml for PHF19 KD at week 8, p=0.07) and bioimaging (Median bioilumisence 2.1x108 p/s for scrambled control vs. 0.8x108 p/s for PHF19 KD at week 8, non-significant). Median OS in mice injected with PHF19 KD cell was substantially longer (66 days) compared to mice subjected to scrambled control cells (54 days), p=0.052. Conclusion- In summary we show that PHF19 is upregulated in malignant plasma cells of MM patients and that PHF19 expression levels increase with advanced MM stages. High PHF19 expression was a marker of adverse prognosis in our total therapy (TT 3-5) cohort. Most importantly, in-vitro and in-vivo functional studies showed that PHF19 has important biological functions in MM. These results suggest that epigenetic regulation through histone methylation, in particular, H3K27 trimethylation, plays a crucial role in MM and the affected downstream pathways should be further elucidated. Disclosures Boyle: Janssen: Honoraria, Other: Travel; Abbvie: Honoraria; Amgen: Honoraria, Other: travel; Takeda: Honoraria, Other: travel; Celgene Corporation: Honoraria, Other: Travel. van Rhee:Kite Pharma: Consultancy; Adicet Bio: Consultancy; Karyopharm Therapeutics: Consultancy; Takeda: Consultancy; Sanofi Genzyme: Consultancy; Castleman Disease Collaborative Network: Consultancy; EUSA: Consultancy. Walker:Celgene: Research Funding.

The Combination of the mTOR Inhibitor Rapamycin and Proteasome Inhibitor Bortezomib Is Synergistic In Vitro in Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3495-3495 ◽  
Author(s):  
Garrett O’Sullivan ◽  
Xavier Leleu ◽  
Xiaoying Jia ◽  
Evdoxia Hatjiharisi ◽  
Hai Ngo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Stromal Cells ◽  
Mtor Inhibitor ◽  
Significant Inhibition ◽  
Synergistic Activity ◽  
Higher Doses

Abstract Background: The PI3K and NFk-B/proteasome pathways are major regulators of survival in Multiple Myeloma (MM). Previous studies have demonstrated clinical efficacy of bortezomib in MM; however, not all patients responded to this agent. mTOR inhibitors have demonstrated significant in vitro and in vivo activity in MM, specifically clinical trials with the mTOR inhibitor CCI-779 (Wyeth) in MM. Therefore, we examined whether inhibition of the PI3K pathway by mTOR and proteasome inhibitors may lead to synergistic activity in MM. Methods: MM cell lines (MM.1S, RPMI, U266, OPM2) were treated with rapamycin 1–5nM (Sigma Aldrich), bortezomib 2.5–10nM (Millenium, MA), or the combination. Cytotoxicity was measured by the MTT assay at 48 hrs; DNA synthesis was measured using thymidine uptake assay; apoptosis was studied using Apo2.7 by flow cytometry, and cell cycle regulation was determined using flow cytometry. To determine whether these agents can overcome the growth advantage conferred by bone marrow stromal cells (BMSCs), we co-cultured cell lines with stromal cells. Normal peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers. Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method, with synergistic activity determined as a combination index (CI) of <1.0. Results: Rapamycin induced dose-dependent cytotoxicity from 0.1nM to 1nM, with an IC50 of 5nM in MM.1S and OPM2. Interestingly, higher doses did not induce further cytotoxicity, confirming that low doses of rapamycin are as effective as higher doses. RPMI and U266 MM cell lines were less sensitive to rapamycin, with 5nM inducing 40% and 20% decrease in survival, respectively. Bortezomib induced significant inhibition of survival in all MM cell lines with an IC50 of 2.5nM, as previously reported. The combination of agents induced significant inhibition of proliferation as compared to each agent alone, specifically with the combination of 5nM rapamycin with 5nM of bortezomib. In the DNA synthesis assay, the combination of bortezomib and rapamycin was significantly cytotoxic compared to each agent alone, specifically at the dose of 5nM rapamycin and bortezomib 2.5nM. The combination of rapamycin 1 to 5 nM and bortezomib 5 to 10 nM were synergistic with a CI index less than 1.0, as in RPMI (CI=0.4) and U266 (CI=0.2) cell lines. The combination of rapamycin and bortezomib at serial concentrations did not trigger cytotoxicity in PBMCs from normal volunteers, indicating significant cytotoxicity in malignant cells, with lack of toxicity in normal PBMCs and suggesting a therapeutic index. The combination of bortezomib and rapamycin demonstrated a significant inhibitory effect on the growth of MM cell lines even in coculture with stromal cells. Cell cycle analysis demonstrated G1 arrest at 24 and 48 hrs in MM.1S cells. Similar results were obtained using primary CD138+ myeloma cells from patients. Conclusion: The combination of rapamycin and bortezomib resulted in synergistic in vitro cytotoxicity in MM cells. These results provide the framework for clinical trials evaluating the combination of CCI-779 and bortezomib in MM.

Validation of the Function of 14-3-3 ζ in Multiple Myeloma (MM)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1369-1369
Author(s):  
Yanyan Gu ◽  
Jonathan L. Kaufman ◽  
Lawrence H. Boise ◽  
Sagar Lonial
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Stable Cell Lines ◽  
Induced Apoptosis ◽  
The Impact

Abstract Abstract 1369 The 14-3-3 protein family includes seven members, β, γ, ε, η, σ, τ and ζ. With over 200 binding partners, 14-3-3 proteins act as integrators of diverse cell signaling pathways and participate in metabolism, cell cycle regulation, survival and apoptosis. 14-3-3ζ has been implicated in many cancers such as hepatocellular carcinoma, gastric cancer, breast cancer, lung carcinoma and lymphoma. However, the role of 14-3-3ζ in MM has not been extensively explored. Preliminary data from an affymatrix GEP profile of normal plasma cells (NPC), MGUS, Smoldering myeloma (SM) or multiple myeloma (MM) demonstrates statistically increased expression of 14-3-3 ζ in the transition between MGUS and SM. Among patients with newly diagnosed symptomatic MM, 14-3-3 ζ expression appears to be higher in the higher risk genetic subsets. These data suggest 14-3-3ζ plays a prominent role in the biology of MM especially among high risk myeloma patients. In order to identify the impact of 14-3-3 ζ signaling on MM proliferation and survival, we developed 14-3-3ζ silenced and over expressing stable cell lines to interrogate the biological role of 14-3-3ζ in MM. Using a library of human MM cell lines, we found that 14-3-3ζ is universally expressed in all MM cell lines examined. Knockdown of 14-3-3ζ significantly inhibits cell growth and proliferation in LP1 and U266 cells, which is partly related to G1 cell cycle arrest. Relevant signaling proteins such as Mcl-1, Bcl2, phospho-Akt and CDK6 decrease after silencing 14-3-3ζ. Furthermore, we performed gene expression profiling of LP1 scrambled and knockdown stable cell lines in order to identify key changes in gene regulation that may be mediated via 14-3-3ζ. The GEP data suggests that 14-3-3ζ is responsible for but not limited to several important signaling pathways, such as glycolysis/gluconeogenesis, p53 Signaling, NRF2-mediated oxidative stress response and death receptor signaling. In addition, we evaluated the effect of 14-3-3ζ expression on the drug sensitivity to commonly used chemotherapeutic compounds in MM treatment, such as bortezomib, etoposide, dexamethasone, melphalan, lenalidomide, doxorubicin and romidepsin. Knockdown 14-3-3ζ sensitizes cells to romidepsin- induced apoptosis, as demonstrated by Annexin V staining and western blot assay for caspase cleavage. However, bortezomib- induced apoptosis is significantly inhibited when 14-3-3ζ is silenced. Bortezomib (5nM)-induced apoptosis decreased from 37% in LP1 cells expressing shRNA with scrambled sequence to 14% in LP1 cells where 14-3-3 ζ is silenced. Moreover, 14-3-3ζ knockdown effectively inhibits bortezomib induced NOXA upregulation, suggesting a possible new molecular mechanism for the effects of 14-3-3ζ in bortezomib mediated apoptosis. Taken together, our work reveals the important biological function of 14-3-3ζ in MM growth, survival and proliferation; the data also provides valuable information for the development of new therapeutic strategies facilitating drug sensitivity and overcoming drug resistance. Disclosures: Kaufman: Millenium: Consultancy; Onyx Pharmaceuticals: Consultancy; Novartis: Consultancy; Keryx: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Onyx: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Millennium Pharmaceuticals, Inc.: Consultancy; Merck: Consultancy.

Simvastatin Induces Death of Multiple Myeloma Cell Lines

2004 ◽  
Vol 52 (5) ◽  
pp. 335-344 ◽  
Author(s):  
Naomi Gronich ◽  
Liat Drucker ◽  
Hava Shapiro ◽  
Judith Radnay ◽  
Shai Yarkoni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cytoplasmic Calcium ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
Rpmi 8226

BackgroundAccumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy.MethodsU266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity.ResultsExposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected.ConclusionsSimvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

Sign in / Sign up

Export Citation Format

Share Document