Hypoxia Reduces CD138 Expression and Induces Immature Phenotype in Myeloma Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3956-3956
Author(s):  
Yawara Kawano ◽  
Shiho Fujiwara ◽  
Naoko Wada ◽  
Hiromichi Yuki ◽  
Hiroaki Mitsuya ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Transcription Factor ◽  
Plasma Cells ◽  
Hypoxic Condition ◽  
Pcr Analysis ◽  
Normoxic Condition ◽  
Rt Pcr ◽  
Down Regulation ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract Abstract 3956 Introduction: Although CD138 expression is a hallmark of plasma cells and myeloma cells, decreased expression of CD138 is occasionally found. We previously reported in the last ASH meeting that 1) CD138 expression decreases in patients with relapsed/progressive disease compared with untreated MM patients and that 2) Patients with low levels of CD138 expression had a worse overall survival compared with patients with high levels of CD138 expression. However, the mechanisms of CD138 down-regulation in myeloma cells are still unclear. It is known that myeloma patient's bone marrow environment is hypoxic (Colla et al. Leukemia. 2010; 24: 1967–1970). It is also reported that tumor progression delivers hypoxic environment to MM cells in vivo (Azab et al. Blood. 2012; 119: 5782–5794). Based upon our and other reports, we hypothesized that CD138 expression may be down regulated by hypoxia. In the present study, we examined changes of CD138 and transcription factor expression in myeloma cells under hypoxic condition. Materials and methods: Two myeloma cell lines (RPMI 8226 and KMS-12-BM) were cultured under normoxic (20% O2) and hypoxic (1% O2) conditions for 24 hrs to 72 hrs. CD138 expression of these cell lines under normoxic and hypoxic conditions were analyzed by flow cytometry. Only viable cells were analyzed by excluding 7-AAD positive dead cells. Real time RT-PCR analysis was utilized to analyze gene expression between normoxic and hypoxic cells. Amounts of IRF4 at protein level were determined with western blotting and flow cytometry. Results: CD138 expression was found to decrease under hypoxic condition compared to normoxic condition in 24 hrs in culture of RPMI 8226 and in 48 hrs in KMS-12-BM when examined with flow cytometry. Real time RT-PCR analysis revealed that CD138 expression was down-regulated under hypoxic condition, indicating that CD138 was down-regulated at transcriptional level. Gene expressions of IRF4, PRDM1 and XBP1, known as plasma cell specific transcription factors, were down-regulated under hypoxic condition compared to those under normoxic condition. Western blot and flow cytometry analysis showed IRF4 was also down-regulated under hypoxic condition. Interestingly, the decreased CD138 expression rendered under hypoxic condition recovered when they were placed under normoxic condition along with an increase of IRF4 expression. Conclusions: We conclude that hypoxia induces down regulation of CD138 in myeloma cells, accompanying with decreased expressions of IRF4, PRDM1 and XBP1. The present data together with our previous finding that reduced CD138 expression is frequently seen in patients with relapsed/progressive diseases suggest that the microenvironment of myeloma cells in patients with relapsed/progressive diseases may be more hypoxic comparing to that at diagnosis. Since IRF4 is a master transcription factor required for the maturation of plasma cells, the hypoxia-induced down-regulation of IRF4 suggests phenotypic shift from mature to immature state, which resembles to the report demonstrating a shift of disease status by hypoxia to more aggressive and immature phenotypes in solid tumors (Axelson et al. Semin Cell Dev Biol. 2005; 16: 554–563). Since IRF4 is also important as a target of IMiDs, further analysis of CD138-negative myeloma cells might contribute not only to better understanding of disease progression but also to drug resistance mechanisms. Disclosures: No relevant conflicts of interest to declare.

scholarly journals RT-PCR analysis of corticotroph-associated genes expression in carcinoid tumours in the ectopic-ACTH syndrome

2006 ◽  
Vol 154 (1) ◽  
pp. 159-166 ◽  
Author(s):  
M Messager ◽  
C Carrière ◽  
X Bertagna ◽  
Y de Keyzer
Keyword(s):  
Transcription Factor ◽  
Molecular Mechanisms ◽  
Ectopic Acth Syndrome ◽  
Pcr Analysis ◽  
Peroxisome Proliferator ◽  
Rt Pcr ◽  
Ectopic Acth ◽  
Peroxisome Proliferator Activated Receptor ◽  
Carcinoid Tumours ◽  
Transcription Factor Genes

Objective: ACTH is frequently produced in non-pituitary tumours, leading to the ectopic-ACTH syndrome, but the molecular mechanisms of its expression remain obscure. This study was aimed at understanding the transcription mechanisms of the ACTH-precursor gene in carcinoid tumours of the lung or thymus. Design: Transcripts coding for a series of corticotroph-associated transcription factor genes were detected, together with markers of the corticotroph phenotype. We studied a series of 41 carcinoid tumours including 15 with proven ectopic-ACTH syndrome. Methods: Specific RT-PCR reactions were designed for each gene including alternatively spliced isoforms. Results: The markers of the corticotroph phenotype were detected in all ACTH-positive tumours. Expression of the Tpit and Pitx1 genes were not restricted to ACTH-positive tumours but were also detected in many ACTH-negative carcinoids. Only a subset of ACTH-negative tumours expressed NAK-1/Nur77, and NeuroD1 expression was detected in <50% of the tumours regardless of their secretory status. The glucocorticoid receptor alpha was detected in every tumour in contrast to its beta isoform detectable in a few tumours only. Chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) and peroxisome proliferator-activated receptor (PPAR) γ2 were expressed in 50% of the tumours of each group whereas PPARγ1 was expressed in almost every tumour. Conclusions: ACTH-positive carcinoids do not share a characteristic expression pattern of the corticotroph-associated transcription factor genes, suggesting that the transcriptional mechanisms of the ACTH-precursor gene differ from those in normal pituitary corticotrophs. Expression of Tpit and Pitx1 genes in most carcinoids suggests that some aspects of the pituitary corticotroph phenotype may belong to general carcinoid differentiation.

scholarly journals Therapeutic Targeting of CCR1 to Prevent Dissemination of Multiple Myeloma Plasma Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3099-3099
Author(s):  
Mara N Zeissig ◽  
Duncan R Hewett ◽  
Krzysztof M Mrozik ◽  
Vasilios Panagopoulos ◽  
Monika Engelhardt ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Disease Progression ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Primary Tumour ◽  
Nsg Mice ◽  
Rpmi 8226 ◽  
Retention Signal

Introduction:Multiple myeloma (MM) disease progression is dependent on the ability of the MM plasma cells (PC) to leave the bone marrow (BM), re-enter the peripheral blood (PB) and disseminate to other BM sites. Previous studies show that expression of CXCL12 by BM stromal cells is crucial for MM PC retention within the BM. However, the mechanisms which overcome this retention signal enabling MM PC egress and dissemination via the PB are poorly understood. Previous studies in haematopoietic progenitor cells have demonstrated that CCL3 overcomes the CXCL12 retention signal to drive mobilisation to the PB (Lord et al. Blood 1995). Here, we examined the role of the CCL3 chemokine receptor CCR1 in driving MM PC dissemination. Methods and results: Initially, we assessed the expression of CCR1 protein on CD138+CD38++CD45loCD19- PC from 28 MM, 8 MGUS and 2 SMM patients by flow cytometry. Results show CCR1 expression is significantly increased in newly diagnosed MM compared with premalignant MGUS and SMM patients (p=0.03; CCR1 MFI mean±SEM, MGUS: 53.0±33.6; SMM: 37.6±8.9 MM: 250.9±71.6). Furthermore, CCR1 expression on PB MM PC positively correlated with PB MM PC numbers (p=0.03; n=11 patients). To identify mechanistically how CCR1 may promote dissemination, the effect of CCL3 on the response to CXCL12 in human myeloma cell lines (HMCL) was assessed in vitro. The migration of RPMI-8226 and OPM2 cells was induced by CCL3 or CXCL12 chemoattractant in a transwell assay. Notably, pre-treatment of RPMI-8226 or OPM2 with CCL3 abrogated migration towards CXCL12 and blocked F-actin remodelling in response to CXCL12 in vitro. These findings suggest that CCL3 can desensitise cells to exogenous CXCL12, providing a potential mechanism facilitating loss of the CXCL12 retention signal. To confirm whether CCR1 is required for driving MM PC dissemination, homozygous CCR1 knockout (KO) cells were generated using a lentiviral CRISPR/Cas9 system in OPM2 cells. CCR1-KO OPM2 cells were confirmed to have no detectable CCR1 expression by flow cytometry and could no longer migrate towards CCL3 in vitro. Empty vector (EV) or CCR1-KO OPM2 MM PC were injected into the tibia of immune-compromised NOD-scidgamma (NSG) mice. After 4 weeks, primary tumour within the injected tibia and disseminated tumour in the PB and the contralateral tibia and femur was assessed by flow cytometry. We found that mice bearing CCR1-KO cells have a 45.5% decrease in primary tumour growth (p=0.008; % GFP+ of total mononuclear cells, EV: 77.2±17.2; CCR1-KO: 42.1±24.4), a 97.8% reduction in PB MM PC (p<0.0001; EV: 1.39±0.7; CCR1-KO: 0.03±0.046) anda 99.9% reduction in BM tumour dissemination (p<0.0001; EV: 49.5±17; CCR1-KO: 0.019±0.013), compared with controls. In a supportive study, CCR1 was expressed in the murine MM cell line 5TGM1 using lentiviral transduction. 5TGM1-CCR1 cells were confirmed to express CCR1 by qPCR and were able to migrate towards CCL3 in vitro. 5TGM1-CCR1 or EV cells were injected into the tibiae of C57BL/KaLwRij mice and allowed to initiate systemic MM disease for 3.5 weeks. Importantly, while 55% of control mice exhibited disseminated tumours, this increased to 92% with CCR1 expression (p<0.0001; n=12/group). These data suggest that CCR1 expression on MM PC may play an important role in MM PC dissemination. To determine whether therapeutic inhibition of CCR1 prevents dissemination, the effect of a small molecule CCR1 inhibitor, CCR1i, was assessed in vivo. OPM2 EV or RPMI-8226 cells were injected into the tibia of NSG mice and, after 3 days, mice were treated with CCR1i (15mg/kg) or vehicle twice daily by oral gavage for 25 days. OPM2-inoculated CCR1i-treated mice had 66.1% lower PB MM PC (p<0.0001; % GFP+ of total mononuclear cells, vehicle: 23.9±7.2; CCR1i: 8.1±3.8) and a 22.1% reduction in BM dissemination (p=0.0002; vehicle: 78.1±4.8;CCR1i: 60.8±7.1) compared with controls. Similarly, CCR1i treatment reduced BM dissemination by 59.6% in RPMI-8226 bearing mice (p<0.0001; % GFP+ of total mononuclear cells, vehicle: 0.86±0.15; CCR1i: 0.26±0.05). This suggests that CCR1 inhibition can slow tumour dissemination in vivo. Conclusion:This study identified CCR1 as a novel driver of MM PC dissemination in vivo, at least in part by overcoming the CXCL12 retention signal. Importantly, this study demonstrated for the first time that targeting CCR1 can be a viable therapeutic strategy to limit dissemination and potentially slow disease progression. Disclosures Croucher: Trovagene: Employment.

ABT-737, an Inhibitor of Bcl-2/Bcl-XL Proteins, Is Cytotoxic in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1589-1589
Author(s):  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
John A. Lust ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Cell Lines ◽  
Plasma Cells ◽  
Annexin V ◽  
Significant Activity ◽  
Rpmi 8226 ◽  
Dose Dependent

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA) Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail. Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

CS1: A Potential New Therapeutic Target for the Treatment of Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3457-3457 ◽  
Author(s):  
Eric D. Hsi ◽  
Roxanne Steinle ◽  
Balaji Balasa ◽  
Aparna Draksharapu ◽  
Benny Shum ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Target Cells ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Effector Cells

Abstract Background: To identify genes upregulated in human memory B and plasma cells, naïve B cell cDNA was subtracted from plasma cell and memory B cell cDNA. One gene that was highly expressed in plasma cells encodes CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family. CS1 was originally identified as a natural killer (NK) cell marker. Monoclonal antibodies (mAbs) specific for CS1 were used to validate CS1 as a potential target for the treatment of multiple myeloma (MM). Methods: Anti-CS1 mAbs were generated by immunizing mice with a protein comprising of the extracellular domain of CS1. Two clones, MuLuc63 and MuLuc90, were selected to characterize CS1 protein expression in normal and diseased tissues and blood. Fresh frozen tissue analysis was performed by immunohistochemistry (IHC). Blood and bone marrow analysis was performed using flow cytometry with directly conjugated antibodies. HuLuc63, a novel humanized anti-CS1 mAb (derived from MuLuc63) was used for functional characterization in non-isotopic LDH-based antibody-dependent cellular cytotoxicity (ADCC) assays. Results: IHC analysis showed that anti-CS1 staining occurred only on mononuclear cells within tissues. The majority of the mononuclear cells were identified as tissue plasma cells by co-staining with anti-CD138 antibodies. No anti-CS1 staining was detected on the epithelia, smooth muscle cells or vessels of any normal tissues tested. Strong anti-CS1 staining was also observed on myeloma cells in 9 of 9 plasmacytomas tested. Flow cytometry analysis of whole blood from both normal healthy donors and MM patients showed specific anti-CS1 staining in a subset of leukocytes, consisting primarily of CD3−CD(16+56)+ NK cells, CD3+CD(16+56)+ NKT cells, and CD3+CD8+ T cells. Flow cytometry of MM bone marrow showed a similar leukocyte subset staining pattern, except that strong staining was also observed on the majority of CD138+CD45−/dim to + myeloma cells. No anti-CS1 binding was detected to hematopoietic CD34+CD45+ stem cells. To test if antibodies towards CS1 may have anti-tumor cell activity in vitro, ADCC studies using effector cells (peripheral blood mononuclear cells) from 23 MM patients and L363 MM target cells were performed. The results showed that HuLuc63, a humanized form of MuLuc63, induced significant ADCC in a dose dependent manner. Conclusions: Our study identifies CS1 as an antigen that is uniformly expressed on normal and neoplastic plasma cells at high levels. The novel humanized anti-CS1 mAb, HuLuc63, exhibits significant ADCC using MM patient effector cells. These results demonstrate that HuLuc63 could be a potential new treatment for multiple myeloma. HuLuc63 will be entering a phase I clinical study for multiple myeloma.

C/EBPβ Is a Critical Factor for Proliferation and Apoptosis in MM Cells by Controlling Transcription Factors Like IRF-4, PAX5 and Blimp1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1506-1506
Author(s):  
Rekha Pal ◽  
Martin Janz ◽  
Deborah Galson ◽  
Suzanne Lentzsch
Keyword(s):  
Transcription Factors ◽  
Western Blot ◽  
Cell Lines ◽  
Plasma Cells ◽  
Annexin V ◽  
Critical Factor ◽  
Myeloma Cell ◽  
Down Regulation ◽  
Protein Levels ◽  
Rpmi 8226

Abstract The development and maturation of plasma cells is dictated by multiple interacting transcription factors (TFs). C/EBPb (NF-IL6) is a TF regulated by IL-6 and has profound effects on the regulation of growth, survival and differentiation of B-cells. Mice deficient in C/EBPb show impaired generation of B lymphocytes suggesting that C/EBPb plays an important role in B lymphopoiesis. In this study we delineated the effect of C/EBPb on transcription factors critical for myeloma cell proliferation by over-expressing and inhibiting C/EBPb in myeloma cells. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226 and H929 were transiently transfected with GFP, C/EBPb (pcNF-IL6), and truncated C/EBPb with a deletion of the internal spII-spII fragment [pcmNF-IL6(Dspl)] by using Bio-Rad Gene Pulser Xcell, followed by G418 selection. A pool of transfected cells was selected and subjected to thymidine incorporation, flow cytometry and western blot analysis. We found that transfection of a truncated form of C/EBPb induced a down-regulation of C/EBPb in MM cell lines (MM.1S, RPMI-8226 and H929) as measured by western blot. Down-regulation of C/EBPβ significantly inhibited proliferation and induced apoptosis of MM cell lines analyzed by annexin V-FITC/PI staining. This was accompanied by a complete down-regulation of the anti-apoptotic protein BCL-2. Further, inhibition of C/EBPb completely decreased IRF-4 expression. In contrast, over-expression of C/EBPb increased protein levels of IRF-4 suggesting that IRF-4 is under control of C/EBPb. IRF-4, which was over-expressed in all our tested MM cells lines, is an essential TF for the generation of plasma cells by regulating TFs like Blimp-1 and PAX-5, which are critical for plasma cell differentiation. Our studies showed that down-regulation of IRF-4 resulted in a complete abrogation of Blimp-1 and PAX-5 suggesting that the expression of these factors is C/EBPb/IRF-4 dependent. In conclusion, our data indicate that C/EBPb is an important key regulator for survival and growth of MM cells. We show for the first time that C/EBPb is a critical regulator upstream of IRF-4. Down-regulation of the C/EBPb and consequently IRF-4 results in complete disruption of the network of TFs necessary for MM growth and survival. Targeting C/EBPb may provide a novel therapeutic approach in the treatment of MM.

IKZF1 (Ikaros) Deletions Are a Hallmark of BCR-ABL1 Positive Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 721-721
Author(s):  
Charles G. Mullighan ◽  
Christopher B. Miller ◽  
Letha A. Phillips ◽  
James Dalton ◽  
Jing Ma ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Cell Lines ◽  
Progenitor Cell ◽  
Blast Crisis ◽  
Dominant Negative ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Snp Arrays ◽  
Progenitor Cell Line

Abstract Using high-resolution SNP arrays and genomic resequencing, we recently reported deletions, translocations, and mutations involving regulators of B cell development in 40% of pediatric B-progenitor ALL (Nature2007;446:758). The most frequently involved genes were PAX5, EBF1, IKZF1 (Ikaros), IKZF3 (Aiolos) and LEF1. Ikaros is a transcription factor required for normal lymphoid development and acts as a tumor suppressor in mice. Focal deletions of IKZF1 were observed in 7 B-progenitor ALL cases, suggesting that the previously reported expression of dominant-negative, non-DNA binding Ikaros isoforms may be caused by genomic IKZF1 abnormalities. We have now extended the analysis to 283 pediatric ALL cases, including B-progenitor ALL with high hyperdiploidy (N=39), hypodiploidy (N=10), rearrangement of MLL (N=23), TCF3-PBX1 (N=17), ETV6-RUNX1 (N=48) and BCR-ABL1 (N=21), as well as cases with low hyperdiploid, normal or miscellaneous cytogenetics (N=75), and T-lineage ALL (N=50). We also examined 22 adult BCR-ABL1 positive ALL cases, 37 acute leukemia cell lines and 49 samples from 23 chronic myeloid leukemia (CML) cases at various stages of disease, including 15 with matched blast crisis samples (12 myeloid, 3 lymphoid). All samples were examined with 500,000 feature Affymetrix SNP arrays (250k Sty and Nsp). The 50k Hind and Xba arrays were also used in 252 ALL cases. Sixty-two (20.3%) ALL cases harboured IKZF1 deletions, including 36 of 43 (83.7%) BCR-ABL1 positive B-ALL cases (76.2% of 21 childhood cases, and 90.9% of 22 adult cases). The deletions were limited to IKZF1 in 25 BCR-ABL1 ALL cases, and in 19 cases deleted an internal subset of IKZF1 exons, most commonly 3–6 (Δ3–6). Remarkably, chronic phase CML samples lacked evidence of IKZF1 deletion, whereas four of 15 matched CML blast crisis samples (66% of lymphoid and 17% of myeloid) had acquired an IKZF1 deletion. The IKZF1 Δ3–6 deletion was also detected in the BCR-ABL1 B-progenitor cell lines BV173, OP1 and SUP-B15, the Δ1–6 deletion in the MYC-IGH/BCL2-IGH B-progenitor cell line 380, and Δ1–7 in the BCR-ABL1 B-progenitor cell line TOM-1. RT-PCR analysis for IKZF1 transcripts demonstrated complete concordance between the extent of IKZF1 deletion and the expression of aberrant Ikaros transcripts lacking internal exons. Importantly, on quantitative RT-PCR analysis and western blotting, expression of the dominant-negative Ik6 transcript and protein, which lacks exons 3–6, was exclusively observed in those cases with IKZF1 Δ3–6, demonstrating that the Ik6 transcript is the result of a specific genetic lesions and not alternative splicing of wild-type IKZF1. Lastly, sequence analysis of the IKZF1 Δ3–6 breakpoints indicated that the deletions arise from aberrant activity of RAG-mediated V(D)J recombination. Taken together, these data demonstrate that deletion of IKZF1, resulting in either haploinsufficiency or the expression of a dominant negative form of the transcription factor, is a central event in the pathogenesis of both pediatric and adult BCR-ABL1 B-progenitor ALL.

Induction Of Proliferation and Survival Of Multiple Myeloma Cells By CD137 Ligand Reverse Signaling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2537-2537
Author(s):  
Chengcheng Fu ◽  
Hui Liu ◽  
Juan Wang ◽  
Ling Ma ◽  
Songguang Ju ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Cell Surface ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Cell Counting ◽  
Myeloma Cells ◽  
Reverse Signaling

Abstract CD137 and its ligand are members of the Tumor Necrosis Factor (TNF) receptor and TNF superfamilies, respectively, regulate cell activation and proliferation of immune system. CD137L, in addition to its ability to costimulate T cells by triggering CD137 receptor, also signals back into antigen presenting cells inducing proliferation, prolonging survival and enhancing secretion of proinflammatory cytokines. The expression of CD137L and its function on multiple myeloma cells is unknown. We identified the constitutive expression of CD137L by flow cytometry on U266, RPMI 8226, LP1, MY5 and KMS-11 of Multiple myeloma (MM) cell lines as high as 96%, 97.5%, 89%, 93% and 94%.But, CD137 expressed on the cell surface was low as 4%, 5%, 1%, 2%, 5% respectively. Now that, CD137L was expressed very strongly on MM cell lines, next, we investigated CD137L expression of MM cells from 85 BM samples of patients seen in the hematological Dept of the First Affiliated Hosp. of Soochow University between January 2012 and June 2013 and diagnosed of active multiple MM, including the patients of newly-diagnosed (n=35), relapsed (n=5) and after 2- 4 prior therapies (n=45). The BM samples were examined using antibodies against CD45RO PE-Cy7, CD138 APC-H7, CD38 FITC and CD137L PE, according to standard protocols for surface staining. Indeed, CD137L protein was expressed by a select group of CD45-CD38++CD138+cells as higher than 95%, the same, CD38 and CD138 are expressed more than 90% of the cells of CD45-CD137L+.There were 22 samples from 11 cases collected before and after treatment and this was further evidence that CD137L molecule was consistently expressed on the MM cell surface. However, CD137L expression was not or hardly detectable on normal plasma cells confirmed by CD45+CD38++CD138+ CD56- CD19+, indicating that CD137L was ectopically expressed by MM cells and probably a specific marker of MM cells. The ectopic CD137L expression was not a mere epiphenomenon but was selected for, what function of it? We hypothesized that it would also act as a growth stimulus for B cell cancers. Then we selected U266-a MM cell line to explore the biological effect of CD137L reverse signaling and its underlying mechanism. As a result, in vitro study, U266 cells(2X105/ml))were cultured plate pre-coated with mAb 1F1 or irrelevant mouse IgG at l ug/ml in PBS and at 400 ul per well of 24-well plate or 80 ul per well of 96-well plate and washed twice after overnight incubation at 4°C. The proliferation and survival of U266 was enhanced by stimulating- CD137L mAb (1F1) than those induced by control mouse IgG by cell counting (4.2 X105/ml VS 3.3 X105/ml), WST-8(1.15 VS 0.81) and CFSE assay (930 VS 991) at incubation for 48h. In addition, the cell cycle analysis showed that CD137L induces proliferation and increases the number of cells in the S phase from 36.1% to 42.5% after 72h incubation. The percentage of apoptosis cells (Annexin V+ and PI+) was 19.6% VS 21.2% with no statistical significance. In order to explore the mechanism of the function of CD137L on MM cells, we surveyed the cytokine profiles during the incubation of U266 cells cultured for 2 days with different stimuli with mAb 1F1 compared with the control group. Intracellular cytokine staining showed that treatment of cells with 1F1 increased the production of IL-6 from 3.8% to 63.9% by Flow cytometry. When neutralizing anti-IL-6 mAb (5 ug/ml) was added to the culture medium, the cells(2X105/ml))were cultured for 48 h in pure medium or plus 10 ng/ml Fc or CD137–Fc protein and the cell proliferation measured by WST-8 was 0.79 VS 0.80 VS 0.72.1F1-induced cell proliferation was effectively inhibited. IL-6 can promote cell proliferation and survival of MM. An increase of these cytokines might explain why CD137L expression could stimulate the proliferation of U266. Finally, the U266 cells were treated with bortezomib and the growth of cells was analyzed by WST-8 assay. It demonstrated that bortezomib could inhibit the function of 1F1 and the inhibition ratio of bortezomib was 22%, 51% and 58% at 24h, 48h and 72h. MM is a B-cell malignancy characterized by the clonal expansion and accumulation of malignant plasma cells in the bone marrow. In our study, CD137L is not only a novel ectopic constitutive marker of MM, but also a promoting proliferation factor. This suggests the possibility that its expression on MM cells may be directly target for immunomodulatory therapy for MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Role for Syntenin-1 in Multiple Myeloma Cell Survival

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1008-1008
Author(s):  
Tyler Moser-Katz ◽  
Catherine M. Gavile ◽  
Benjamin G Barwick ◽  
Sagar Lonial ◽  
Lawrence H. Boise
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Cell Survival ◽  
Cell Lines ◽  
Plasma Cells ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract Multiple myeloma is the second most common hematological malignancy in the U.S. with an estimated 30,700 new diagnoses in 2018. It is a clonal disease of plasma cells that, despite recent therapeutic advances, remains incurable. Myeloma cells retain numerous characteristics of normal plasma cells including reliance on survival signals in the bone marrow for long term viability. However, malignant transformation of plasma cells imparts the ability to proliferate, causing harmful bone lesions in patients, and in advanced stages independence of the bone-marrow microenvironment. Therefore, we are investigating the molecular mechanisms of myeloma cell survival that allow them to become extramedullary. We identified syntenin-1 (SDCBP) as a protein involved in myeloma cell survival and a potential therapeutic target. Syntenin-1 is an adapter protein that has been shown to regulate surface expression of several transmembrane proteins by binding with membrane phospholipids and mediating vesicular trafficking of proteins throughout the cell. Syntenin-1 regulates the surface expression of CD138, a plasma/myeloma cell marker. Syntenin-1 has been shown to regulate apoptosis in numerous cancer cell lines including breast cancer, glioma, and pancreatic cancer but its role in multiple myeloma survival has not been studied. To determine if syntenin-1 expression has an effect on myeloma cell survival, we utilized the CoMMpass dataset (IA12), a longitudinal study of myeloma patients that includes transcriptomic analysis throughout treatment. We found that patients with the highest expression of syntenin-1 mRNA (top quartile) had significantly worse overall survival, progression-free survival, and a shorter response duration than those in the bottom quartile of expression. To determine if syntenin-1 has a role in myeloma cell survival, we used short hairpin RNA to knock down syntenin-1 (shsyn) in RPMI 8226 and MM1.s myeloma cell lines. We then determined the amount of cell death using Annexin-V staining flow cytometry four days following lentiviral infection. We found increased cell death in syntenin-1-silenced cells compared to our empty vector control in both RPMI 8226 (control=42.17%, shsyn=71.53%, p=0.04) and MM1.s cell lines (control=8.57%, shsyn=29.9%, p=0.04) suggesting that syntenin-1 is important for myeloma cell survival. Syntenin-1 contains two PDZ domains that allow it to bind to receptor proteins via their corresponding PDZ-binding motifs. We therefore wanted to look at correlation of syntenin-1 expression with CD138 and CD86, two PDZ-binding domain containing proteins expressed on the surface of myeloma cells. Using the CoMMpass dataset, we found patients with high expression of syntenin-1 had a median expression of CD86 that was twice as high as the total population (P<0.0001) while syntenin-1-low patients expressed CD86 at levels that were half as much as the population (P<0.0001). In contrast, there was no clear relationship between syntenin-1 and CD138 mRNA expression. Indeed if one takes into account all patients, there is a positive correlation between CD86 and syntenin-1 expression (r=0.228, P<0.0001) while there is a negative correlation between CD138 and syntenin-1 (r=-0.1923, P<0.0001). The correlation with CD86 but not CD138 suggests a previously undescribed role for syntenin-1 in myeloma cells. Our lab has previously shown that expression of CD86 is necessary for myeloma cell survival, and signals via its cytoplasmic domain to confer drug resistance. Silencing syntenin-1 results in a decrease in CD86 surface expression. However, there is no change in CD86 transcript or total cellular CD86 protein levels in our shsyn treated cells. Moreover, knockdown of CD86 resulted in increased protein expression and transcript levels of syntenin-1. Taken together, these data suggest that syntenin-1 may regulate CD86 expression on the cell surface. Our data supports a novel role for syntenin-1 in myeloma cell viability and as a potential regulator of CD86 surface expression. The role of syntenin-1 has not previously been explored in multiple myeloma and determining its molecular function is warranted as it may be an attractive target for therapeutic treatment of the disease. Disclosures Lonial: Amgen: Research Funding. Boise:AstraZeneca: Honoraria; Abbvie: Consultancy.

Primary Plasma Cells Expressing CD52 Are Efficiently Targeted In Vivo by Alemtuzumab.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3460-3460
Author(s):  
Carmelo Carlo-Stella ◽  
Massimo Di Nicola ◽  
Paolo Longoni ◽  
Loredana Cleris ◽  
Marco Milanesi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Plasma Cells ◽  
Therapeutic Potential ◽  
Significant Proportion ◽  
Scid Mice ◽  
Rank Test ◽  
Pcr Analysis ◽  
Significant Prolongation

Abstract Salvage therapy options for multiple myeloma (MM) patients relapsing following autologous stem cell transplantation remain limited. New treatments targeting the malignant plasma cell (PCs) are therefore needed. Alemtuzumab is a humanized monoclonal antibody to CD52 capable of destroying CD52+ cells by antibody-mediated cellular cytotoxicity and complement fixation. In order to evaluate the therapeutic potential of alemtuzumab for MM patients, we initially examined CD52 expression on primary malignant marrow PCs as well as a panel of MM continuous cell lines (RPMI-8226, KMS-11, OPM-2, U-266, LP-1, ARH-77). In addition, the anti-myeloma activity of alemtuzumab was evaluated in vivo in a xenotransplant model of MM in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. PCs were enriched from the marrow of MM patients (n = 40) according to CD138 expression. By using an immunomagnetic technique (Miltenyi Biotec, Germany, EU), highly purified CD138+ cells (median purity = 93%; median recovery = 55%) were obtained and further characterized by 3-color (CD138/CD45/CD52) flow cytometry. Expression of CD52 on CD138-enriched cells was detected in 28/40 (70%) of MM patients, with a median of 95% PCs expressing CD52. Detection of CD52 was equally evident on CD138+CD45+ and CD138+CD45− PCs (P ≥ 0.05). Similarly to what observed for primary CD138+ cells, MM cell lines showed a rather heterogeneous CD52 expression, ranging from dim (KMS-11, OPM-2) to bright positivity (LP-1, ARH-77). Both on primary PCs and MM cell lines, expression of CD52 mRNA by quantitative PCR analysis strongly correlated with CD52 antigen detection by flow cytometry. The in vivo activity of alemtuzumab was evaluated in a xenotransplant model of MM in NOD/SCID mice. Mice were inoculated intravenously with KMS-11 cells (0.5 x 105 per mice) and were treated with alemtuzumab (3 x 1 mg/mouse, subcutaneously, days 2, 5, 7). All placebo-treated mice (n = 15) died, whereas mice treated with alemtuzumab (n = 15) showed a significant prolongation of median survival (P ≤0.009 by log rank test) and 27% of them were alive and well at 120 days. No mice experienced any apparent treatment-related toxicity. According to these data, we conclude that: (1) CD52 is expressed on the plasma cells of a significant proportion of MM patients; (2) alemtuzumab has a strong antitumor activity in vivo on CD52-positive MM cell lines; (3) alemtuzumab might have therapeutic potential in a subset of MM patients.

High Levels of Soluble Immunoregulatory Receptors in Patients with WaldenströM’s Macroglobulinemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4881-4881 ◽  
Author(s):  
Zachary R. Hunter ◽  
Andrew R. Branagan ◽  
Daniel Ditzel Santos ◽  
Olivier Tournilhac ◽  
Evdoxia Hatjiharissi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Mast Cells ◽  
Immune Responses ◽  
Pcr Analysis ◽  
P Value ◽  
Rt Pcr ◽  
T Cell Immune Responses ◽  
Immunoregulatory Receptors ◽  
Cell Disorder

Abstract CD25, CD27, CD30 and CD40 are receptors for IL-2, CD70, CD153 and CD154, respectively, which provide important signaling for both B- and T-cell immune responses. We therefore examined the sera of patients with Waldenstrom’s macroglobulinemia (WM), a B-cell disorder characterized by excess IgM secreting bone marrow lymphoplasmacytic cells (LPC) for the presence of soluble CD25, CD27, CD30, CD40, IL-2, CD153, and CD154. Sera were from patients with active disease and off-therapy. Sera from healthy age matched donors (HD) was used for controls. The results of these studies were as follows: WM ELISA Results HD ELISA Results p-value n= median range n= median range sCD25 pg/ml 2.647x10−6 41 3418.99 3–19756.2 20 573.6 184.96–891.03 IL-2 pg/ml 0.72385 41 22.96 3–76.83 20 11.07 5.64–64.7 sCD27 U/ml 2.4727x10−7 26 7.45 0–19.42 10 0 0–2.78 sCD70 ND ND ND ND ND ND ND sCD30 U/ml 0.00088 25 89.25 0–550.81 15 39.12 0–135.25 sCD153 ng/ml 0.68388 25 8.18 0–43.8 15 5.65 0–24.82 sCD40 pg/ml 0.02484 25 101.39 11.76–584.99 11 50.73 0–150.5 sCD154 pg/ml 0.81317 55 949.58 92–4973.16 17 1078.03 351.88–2323.38 Considering our recent studies demonstrating a role for mast cells (MC) in supporting WM cell growth (JCO 2004 22:571S), we examined bone marrow LPC along with MC from WM patients for expression of CD25, CD27 and CD40 and/or their ligands by flow cytometry and RT-PCR analysis. The results of these studies are as follows: Flow Cytometry RT-PCR WM MC WM MC CD25 4/10 (40%) 2/7 (29%) 7/9 (78%) 7/15 (47%) IL-2 ND ND ND ND CD27 5/12 (42%) 2/8 (25%) 7/7 (100%) 4/7 (57%) CD70 6/6 (100%) 10/11 (91%) 7/7 (100%) 2/7 (29%) CD30 1/21 (4.7%) ND ND ND CD153 3/7 (43%) 12/13 (92%) 2/2 (100%) 9/11 (82%) CD40 25/30 (83%) ND ND ND CD154 2/3 (67%) 9/13 (69%) 7/9 (78%) ND The above studies demonstrate high levels of circulating immunoregulatory receptors, and expression of these receptors and/or their ligands on WM tumor and mast cells. Studies addressing their role in immune dysregulation in WM are underway.

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