scholarly journals Ablation of CD28-86 Signaling Results in Induction of Both Caspase-Dependent and Caspase-Independent Cell Death in Myeloma Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4726-4726
Author(s):  
Catherine M Gavile ◽  
Ajay K. Nooka ◽  
Sagar Lonial ◽  
Kelvin P Lee ◽  
Lawrence H. Boise
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Family Members ◽  
Myeloma Cell ◽  
Chemotherapeutic Agents ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Survival Mechanisms ◽  
Survival Signals

Abstract Multiple myeloma is a disease of long-lived plasma cells (LLPCs), and is the 2nd most common hematologic malignancy. In recent years, pharmacologic advances have led to an increase in median and overall survival rates. However, the disease remains incurable for the majority of patients, and research on the underlying survival mechanisms of myeloma cells is relevant for discovering new therapeutic approaches that could eventually lead to a cure. Myeloma cells retain most of the physiological characteristics of their normal counterpart – the LLPC. They secrete antibodies, express CD138, and home and reside in the bone marrow, where they are heavily reliant on growth and survival signals from the stromal microenvironment. Recent reports have shown that the CD28-86 costimulation pathway is important for the generation and survival of LLPCs. Consistent with a pro-survival function, previous studies have demonstrated that CD28 and CD86 high expression are poor prognostic indicators for myeloma patients. Additionally we have shown that CD28 signaling mediates resistance to different chemotherapeutic agents. To better understand the role of CD28 and CD86 in myeloma we have been studying the effects of loss of expression or blockade in myeloma cell lines and patient samples. We have shown that myeloma cells also require CD28-86 signaling for their survival, as knockdown of either CD28 or CD86 via shRNAs, or blockade with CTLA4Ig (Abatacept), led to cell death in 5 myeloma cell lines and 1 patient sample. We have also shown that CD28-86 signaling regulates expression of integrins (β7, β1) that play important roles in cell-cell or cell-matrix interactions that facilitate cell growth and survival. Taken together, our previous work indicates that the CD28-86 signaling pathway plays an important role in maintaining myeloma cell viability. Interestingly, our data indicate that CD86 relays a survival signal that is different from its function as a CD28 ligand. Overexpression of an shRNA-resistant CD86 (CD86FLm) protected against CD86 silencing, while overexpression of CD86TLm (where the intracellular domain of CD86 has been deleted) does not, indicating that the cytoplasmic tail of CD86 plays a role in myeloma cell survival. In order to determine the survival mechanisms mediated by this signaling pair, we investigated different pathways known to protect myeloma cells from pro-apoptotic signals.We first demonstrated that exogenous IL-6, a myeloma growth and survival factor, cannot protect against cell death from CD28 or CD86 silencing, suggesting that the CD28-CD86 pathway is distinct from IL-6 signaling and provides survival signals that are complementary to IL6 receptor signaling. In contrast, overexpression of pro-survival Bcl-2 family members protects against cell death induced by silencing of CD28 and CD86. However, when we performed expression analyses (RNA-seq, pRT-PCR and Western blot), no consistent significant changes were observed in any of the Bcl-2 family members following CD28 or CD86 knockdown. Since Bcl-2 proteins can inhibit both apoptotic and non-apoptotic forms of cell death (e.g. autophagy), we determined if the cell death was caspase dependent. Caspase-3 is activated by CD28 or CD86 silencing or CTLA4Ig treatment. However pan-caspase inhibitors Boc-D-FMK or QVD-Oph can only partially protect against this cell death despite demonstrating a complete blockade of caspase-3 cleavage. Overall, our data show that cell death induced upon ablation of CD28-86 signaling is pleiotropic, as it appears to be both caspase-dependent and caspase-independent. We will present data on the mechanism of non-apoptotic death (autophagy or necrosis). Preliminary data indicates that autophagy is activated by CD28/CD86 silencing. Our data suggest that blocking the CD28-86 pathway may be a viable therapeutic addition to current regimens since it induces myeloma cell death through multiple mechanisms and therefore may not be susceptible to drug resistance that is associated with relapsed/refractory disease. Disclosures No relevant conflicts of interest to declare.

Interfering with Arginine Metabolism As a New Treatment Strategy for Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3005-3005
Author(s):  
Bjoern Jacobi ◽  
Lea Stroeher ◽  
Nadine Leuchtner ◽  
Hakim Echchannaoui ◽  
Alexander Desuki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Xenograft Model ◽  
Myeloma Cell ◽  
Intracellular Protein ◽  
Myeloma Cells ◽  
Induction Of Apoptosis

Abstract Introduction Starvation of tumor cells from the amino acid arginine has recently gained particular interest because of the downregulation of the rate-limiting enzyme argininosuccinate synthethase 1 (ASS1) in various cancer entities. ASS1-deficient cells cannot resynthesize arginine from citrulline and are therefore considered arginine auxotrophic. The arginine depleting enzyme arginine deiminase (ADI-PEG20, Polaris Pharmaceuticals) is currently tested in phase I-III clinical trials for different arginine auxotrophic cancers. The natural arginine analogue canavanine can compete with arginine for arginyl-tRNA-binding sites and consequently be incorporated into nascent proteins instead of arginine. Canavanine could therefore potentially further disturb intracellular protein homeostasis, especially under arginine deprivation. The sensitivity of myeloma cells towards arginine depletion strategies has not been analyzed so far. Methods Human myeloma cell lines and CD138-sorted primary human myeloma cells from patient bone marrow were screened for ASS1 expression by western blotting (WB). The cells were cultured in arginine free medium and assessed for proliferation and metabolic activity (CFSE/MTT assays), apoptosis (caspase-3 cleavage) and cell death (annexinV/propidium iodide). Canavanine was supplied in both arginine-sufficient and -deficient conditions. The level of intracellular protein stress was determined by WB and/or flow cytometry analysis for ubiquitinated proteins, phosphorylated eukaryotic initiation factor 2α (peIF2α) and the spliced isoform of the X-Box binding protein 1 (Xbp1s). Repetitive ADI-PEG20 ± canavanine application i.p. were tested in vivo in an U266 myeloma xenograft model in NOD/SCID/IL2Rcg-/- (NSG) mice. Arginine and canavanine levels in plasma were determined by HPLC. Tumor growth was measured, mice were assessed for survival, weight and side effects. Tumor tissues were analyzed for caspase-3 cleavage and Ki67 expression by immunohistochemistry. Results 5 of 6 myeloma cell lines were negative for ASS1. Also, ASS1 was either not or only weakly expressed in the majority of primary CD138+ myeloma patient samples. Arginine starvation induced an arrest of cell proliferation and/or metabolic activity of primary myeloma cells and myeloma cell lines after 18-24 h. Addition of citrulline could only rescue ASS1 positive myeloma cells due to the intracellular resynthesis of arginine. Arginine starvation alone led to delayed induction of apoptosis (e.g. 35% cell death of NCI-H929 cells after 72 h of treatment). Addition of 100 mM canavanine strongly increased cell death specifically in the context of arginine deficiency (e.g. cell death in NCI-H929 cells: 87% after 24 h, 100 % after 48h) while it was non-toxic and had no effect on cell viability under physiological arginine conditions. Co-application of canavanine induced ubiquitination of cellular proteins and led to the prolongation of a fatal unfolded protein response (UPR) as measured by markedly elevated Xbp1s levels. Prolonged UPR ultimately led to the induction of apoptosis as reflected by annexin V binding and caspase-3 cleavage. In an U266 myeloma NSG xenograft model, systemic arginine depletion by ADI-PEG20 suppressed tumor growth in vivo and significantly prolonged median survival of mice when compared with the control group (22±3 vs. 15±3 days). Canavanine treatment alone had no influence on viability (13±0 days). However, the combination of ADI-PEG20 and canavanine demonstrated the longest median survival (27±7 days). Histological examination of explanted tumors showed the highest rates of caspase-3 cleavage in the ADI-PEG20/canavanine group. Conclusion Myeloma cells are mostly arginine auxotrophic and can be selectively targeted by arginine starvation. Combination of arginine depletion with the arginine analogue canavanine leads to a highly efficient and specific tumor cell eradication and should be further optimized in multiple myeloma preclinical models. Disclosures Bomalaski: Polaris Pharmaceuticals Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

TRAILR Triggering by Human Antibodies Induces Apoptosis through an Early Cleavage of Mcl-1 and Caspase 3 in Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3397-3397
Author(s):  
Catherine Pellat-Deceunynck ◽  
Patricia Gomez-Bougie ◽  
Sylvanne Daniels ◽  
Alexandrine Geffroy-Luseau ◽  
Regis Bataille ◽  
...  
Keyword(s):  
Cell Lines ◽  
Caspase 3 ◽  
Chromosomal Translocation ◽  
Myeloma Cell ◽  
Caspase 8 ◽  
Early Cleavage ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Human Antibodies

Abstract We investigated TRAILR expression and sensitivity of myeloma cells in vitro. This study was done using a panel of 20 myeloma cell lines that are representative of primary myeloma cells (14q chromosomal translocation, IL-6 dependency, phenotype, oncogenes mutation). TRAILR were stimulated with agonistic human antibodies directed against either TRAIL-R1/DR4 (HGS-ETR1, mapatumumab) or TRAIL-R2/DR5 (HGS-ETR2), provided by Human Genome Sciences, Rockville, MD. This approach allowed us to analyze the contribution of each receptor separately. We show that a wide majority of cell lines, 16 of 20 were killed upon either TRAIL-R1 or R2 stimulation in the presence or absence of IL-6. However, 4 cell lines were resistant to HGS-ETR1 and 6 to HGS-ETR2 and 3 to both. Activation of both caspase 8 and Bid has been extensively described as being associated with TRAIL response. Indeed, we observed an activation of both caspase 8 and Bid. Cleaved molecules were detected 6 to 18h after antibody addition but after detection of cellular apoptosis. However, we show that Mcl-1L, a key molecule for myeloma survival, was downregulated and cleaved as soon as 3h after Ab addition. The cleaved form of Mcl-1 has been shown to behave like a proapoptotic molecule. Since caspase 3 has been reported to cleave Mcl-1, we looked at caspase 3 activation. Indeed, we observed that caspase 3 cleavage occured early and concomitantly to the one of Mcl-1. Our data show that in a wide majority of myeloma cell lines (80%) TRAILR triggering induces massive apoptosis that was not prevented by IL-6, the major myeloma cell growth and survival factor. Moreover, apoptosis induced upon TRAILR triggering was fully correlated to an early cleavage of both caspase 3 and Mcl-1 and to a delayed one of both caspase 8 and Bid.

TP53 Positively Regulates Cell Death through TRAILR2 (DR5) but Negatively through TRAILR1 (DR4) In Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 136-136
Author(s):  
Sylvanie Surget ◽  
Patricia Gomez-Bougie ◽  
David Chiron ◽  
Robin Humphreys ◽  
Philippe Moreau ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Caspase 8 ◽  
Myeloma Cells ◽  
Trail Receptors ◽  
Tp53 Status

Abstract Abstract 136 Mapatumumab and lexatumumab are human antibodies that bind and activate death receptors TRAILR1/TNFSF10A/DR4 and TRAILR2/TNFSF10B/DR5, respectively. Treatment of primary myeloma cells and myeloma cell lines with these mAbs induced cell death. Mapatumumab induced cell death more effectively than lexatumumab in a panel of 30 human myeloma cell lines (HMCLs). Interestingly, sensitivity to mapatumumab and lexatumumab was mutually exclusive and related to TP53 status (p=0.006). Indeed, wildtype TP53 HMCLs (n=9) were sensitive to lexatumumab (mean of death 40%) but resistant to mapatumumab (mean of death 7%). In contrast, abnormal (n=21) TP53 HMCLs were resistant to lexatumumab (mean of death 7%) but sensitive to mapatumumab (mean of death 44%). Of note, killing by lexatumumab was correlated to TRAILR2 expression while no correlation was found for TRAILR1 expression and lapatumumab killing. Transcriptomic analysis of 30 HMCLs revealed that HMCLs with abnormal TP53 status underexpressed 4 well-known p53 target genes, MDM2, CDKN1A, Bax and TRAILR2 (p<0.01). To activate p53 pathway in myeloma cells, we used melphalan at low doses. Melphalan treatment of wildtype HMCLs, but not of TP53 abnormal HMCLs, increased TRAILR2 expression and cell death mediated by lexatumumab. In contrast, melphalan did not alter TRAILR1 expression or mapatumumab-induced killing, suggesting that TRAILR2 but not TRAILR1 is a p53 target in myeloma cells. Silencing TP53 significantly increased mapatumumab apoptosis (>50% p<0.01). In good agreement with Bax underexpression in TP53 abnormal HMCLs, extensive silencing of key molecules of both extrinsic (caspase 8, caspase 3) and intrinsic pathways of apoptosis (caspase 9, Bid, Bim, Bax) showed that mapatumumab killing was dependent on the extrinsic pathway of apoptosis only: only silencing of caspase 8 or caspase 3 inhibited mapatumumab killing. Altogether, these data show that killing through TRAIL receptors is differentially regulated by p53 in myeloma cells, positively for TRAILR2 but negatively for TRAILR1. Interestingly, myeloma cells with an abnormal p53 that are more resistant to all drugs are more sensitive than wt ones to killing through TRAILR1 making this pathway very attractive for p53 deficient myeloma cells. Disclosures: No relevant conflicts of interest to declare.

Autophagy Is a Key Myeloma Survival Pathway That Can Be Manipulated Therapeutically to Enhance Apoptosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4083-4083
Author(s):  
Lauren I Aronson ◽  
Emma L Davenport ◽  
Serena G Giuntoli ◽  
Muralikrishnan Srikanth ◽  
Emma Smith ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Mtor Pathway ◽  
Mtor Inhibition ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Survival Pathway ◽  
In Cells

Abstract Abstract 4083 In normal cells, autophagy is up-regulated under conditions of stress ensuring cell survival, suggesting that if it is inhibited cell death will ensue. In some cancer settings however it has been shown to enhance cell death. Therefore if autophagy is to be a target for anti-cancer therapy, it is important to determine whether to promote or inhibit the process. Cellular entry to autophagy can be manipulated via the PI3K/AKT/mTOR pathway, a pathway known to be important for myeloma cell growth and survival. In this study we investigated the effects of PI-103, a dual Class I PI3K and mTOR inhibitor, which activates autophagy, and used it as a tool to investigate the interaction of autophagy with other myeloma therapies. As the PI3K/AKT/mTOR pathway has been suggested to be a central pathway controlling entry into autophagy, we first determined the basal expression of key members of the pathway in a panel of myeloma cell lines. PI3K alpha, beta delta and gamma isoforms were expressed to varying degrees and constitutive activation of the pathway, (pAKT or pMTOR), was seen in the majority of cell lines. As predicted, treatment with PI-103 induced autophagy in myeloma cells as demonstrated by an increase in cellular inclusions staining positively with acridine orange, cleavage of the autophagosome marker, LC3, and a decrease in p62. PI-103 was shown to inhibit proliferation of all the myeloma cell lines and patient cells tested to varying degrees, although 100% growth inhibition was not seen. Bone marrow stromal cells were unaffected. The main mode of action of PI-103 was autophagy activation, and in keeping with this the extent of cell death measured by Annexin V/PI binding and trypan blue exclusion, was minimal. Cell cycle analysis demonstrated an increase in G0-G1 phase. The unfolded protein response (UPR) is important in myeloma cells and UPR activation and autophagy have been reported to be interlinked. Following exposure to PI-103, splicing of XBP1 mRNA to its active form, XBP1s, was seen, CHOP and ATF4 mRNA levels were also increased, consistent with activation of at least two branches of the UPR in response to PI3K/mTOR inhibition and autophagy induction. As myeloma cells activate autophagy as a pro-survival pathway following PI3-kinase inhibition, we were interested to understand the effect of blocking autophagy in this context. When PI-103 was combined with the autophagy inhibitor, Bafilomycin greatly enhanced apoptosis was seen. This increased apoptosis was seen in cells constitutively expressing p-AKT, with a complete loss of both phospho- and total levels of AKT and mTOR, an increase in the cleaved forms of caspase 3 and Bcl2, and massive activation of the IRE1 and PERK branches of the UPR. This effect was not seen in cells lacking p-AKT, a phenomenon described as ‘context-dependent oncogene addiction' suggesting that measurement of p-AKT may be a useful predictive marker for response to joint PI3K/autophagy inhibition. Importantly the pro-apoptotic effects of the combination are not overcome by the presence of bone marrow cytokines, a more representative model of the physiological situation in vivo. In conclusion our data highlights the interplay between known myeloma growth and survival pathways and autophagy and suggests that combining PI3K inhibitors, with agents that target autophagy, may be beneficial for the treatment of myeloma, particularly in >50% of patients that express p-AKT. Disclosures: No relevant conflicts of interest to declare.

Cell death provoked by loss of interleukin-3 signaling is independent of Bad, Bim, and PI3 kinase, but depends in part on Puma

Blood ◽  
2006 ◽  
Vol 108 (5) ◽  
pp. 1461-1468 ◽  
Author(s):  
Paul G. Ekert ◽  
Anissa M. Jabbour ◽  
Anand Manoharan ◽  
Jacki E. Heraud ◽  
Jai Yu ◽  
...  
Keyword(s):  
Cell Death ◽  
Family Members ◽  
Pi3 Kinase ◽  
Minor Role ◽  
Growth And Survival ◽  
Interleukin 3 ◽  
Dependent Cell ◽  
Induced Apoptosis ◽  
A Minor ◽  
Survival Signals

Growth and survival of hematopoietic cells is regulated by growth factors and cytokines, such as interleukin 3 (IL-3). When cytokine is removed, cells dependent on IL-3 kill themselves by a mechanism that is inhibited by overexpression of Bcl-2 and is likely to be mediated by proapoptotic Bcl-2 family members. Bad and Bim are 2 such BH3-only Bcl-2 family members that have been implicated as key initiators in apoptosis following growth factor withdrawal, particularly in IL-3-dependent cells. To test the role of Bad, Bim, and other proapoptotic Bcl-2 family members in IL-3 withdrawal-induced apoptosis, we generated IL-3-dependent cell lines from mice lacking the genes for Bad, Bim, Puma, both Bad and Bim, and both Bax and Bak. Surprisingly, Bad was not required for cell death following IL-3 withdrawal, suggesting changes to phosphorylation of Bad play only a minor role in apoptosis in this system. Deletion of Bim also had no effect, but cells lacking Puma survived and formed colonies when IL-3 was restored. Inhibition of the PI3 kinase pathway promoted apoptosis in the presence or absence of IL-3 and did not require Bad, Bim, or Puma, suggesting IL-3 receptor survival signals and PI3 kinase survival signals are independent.

Nocodazole Induces Myeloma Cell Death by Disrupting the Microtubular Network, Resulting in G2/M Arrest

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3673-3673
Author(s):  
Rentian Feng ◽  
Jorge A Rios ◽  
Markus Mapara ◽  
Suzanne Lentzsch
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Caspase 8 ◽  
Inhibition Rate ◽  
Growth And Survival ◽  
Microtubular Network

Abstract Patients with relapsed multiple myeloma (MM) previously treated with bortezomib and lenalidomide often fail to respond to further therapies. To identify potential new treatment approaches for MM, we used Luminex technology to screen a library of 1,120 compounds provided by the Multiple Myeloma Research Foundation. By multiplex cytokine array, we identified benzimidazoles including the anthelmintics mebendazole, fenbendazole, albendazole, nocodazole and pyrvinium pamoate, as inhibiting the production of cytokines essential for MM cell growth and survival, such as IL-6 (inhibition rate 40–70%), MIP-1α (inhibition rate 65–75%), VEGF (inhibition rate 75%), and soluble IL-6R (inhibition rate 40–52%). Consequently, these anthelmintics demonstrated dose-dependent inhibition of myeloma cell (RPMI-8226, H929, U266 and MM1S) proliferation. The lead compound, nocodazole, caused nuclear fragmentation and caspase-8 activation in MM cell lines and primary CD138+ cells in dose- and time-dependent fashion (IC50: 30–60 nM). Importantly, growth and survival signals provided by bone marrow stromal cells in bone marrow co-cultures failed to protect MM cells from nocodazole-induced cell death. In the apoptotic cells, caspase-8 was more activated than caspase-9, suggesting that mitochondrial signaling is not a major apoptotic pathway. Cell cycle analysis indicated that G2/M cell cycle arrest reached a peak at 17 hr. Sub-G1 proportion was strongly increased after treatment for 24 hr in all tested cell lines. Electron microscope (EM) and nuclear staining studies consistently showed the accumulation of metaphase cells, and morphologic elongation at 7 hr, at which time G2/M arrest was obvious. Most of the elongated cells had only one nucleus, suggesting that they failed to progress to mitosis due to overall microtubular network disarray. We conclude that nocodazole exposure induced microtubular network disarray with cell elongation, and G2/M arrest with a late stage mitotic block resulting in cell death. Benzimidazoles including nocodazole, traditionally used as antihelmintic drugs, have shown antitumor activity against hepatocellular, lung and adrenocortical carcinoma, and melanoma. In our study, we identified the anthelmintic compound nocodazole as a new anti-myeloma agent. Nocodazole warrants further investigation for its anti-MM effects in vitro and in vivo.

Serum/Glucocorticoid-Regulated Kinase 1 (SGK1) Is a Prominent Target Gene of the Transcriptional Response to Cytokines In Multiple Myeloma and Supports the Growth of Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1915-1915
Author(s):  
Unn-Merete Fagerli ◽  
Thorsten Stühmer ◽  
Toril Holien ◽  
Randi Utne Holt ◽  
Ove Bruland ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factors ◽  
Cell Lines ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Transcriptional Response ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Stat3 Phosphorylation

Abstract Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

CD28 and CD86 Are Necessary for Myeloma Cell Survival.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2946-2946
Author(s):  
Catherine M Gavile ◽  
Jayakumar R Nair ◽  
Kelvin P Lee ◽  
Sagar Lonial ◽  
Lawrence H. Boise
Keyword(s):  
T Cells ◽  
Growth Factor ◽  
Cell Survival ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Growth And Survival

Abstract Abstract 2946 Multiple myeloma (MM) is a hematologic malignancy characterized by the aberrant proliferation of plasma cells. Myeloma cells retain most of the physiological characteristics of their normal counterpart – the long-lived plasma cell. Myeloma cells secrete immunoglobulin and reside in the bone marrow, where they rely heavily on interactions with the stroma for survival signals. While recent advances in therapeutics have led to an increase in median survival post-diagnosis, the disease remains incurable. Understanding the pathways which mediate growth and survival of these cells will help in identifying new targets that can potentially further improve patient outcomes. CD28 is a receptor better known for its role in T-cell signaling through interaction with its ligands, CD80 or CD86. Interaction between CD28 on T-cells and CD80/86 on antigen-presenting cells leads to survival and proliferation of T-cells. Recent work has shown that the CD80/86-CD28 pathway also plays an important role in normal plasma cell generation and survival. Interestingly, high expression of CD28 and CD86 are poor prognostic markers for myeloma patients. Previous work has shown that CD28 activation provides survival signals for myeloma cells in growth-factor deficient conditions. It has also been shown that CD28 on the myeloma cell interacts with CD80/86 on the dendritic cell, which induces secretion of IL-6 (by the DC), an important myeloma growth factor. However, it is not known if CD28 or CD86 play a role in steady state growth and survival of myeloma cells. In order to determine the role of each of these 2 molecules in myeloma physiology, we knocked-down either CD28 or CD86 on the myeloma cell via lentivirus-mediated shRNAs. We found that knockdown of CD86 leads to apoptosis in 3 myeloma cell lines (RPMI8226, MM1.s, and KMS18). Four days after infection with the lentivirus containing shCD86, 45.7±4.9 and 60.3±4.6 percent control apoptosis was observed in RPMI8226 and MM1.s respectively, while less death was observed in KMS18 (17.6±1.6). CD28-knockdown resulted in apoptosis as well (24.9±4.3 for RPMI8226, 26.8±4.1 for MM1s, 21.8±3.8 for KMS18, percent control apoptosis). Consistent with these findings, we were unable to establish a myeloma cell line with stable knockdown of either CD28 or CD86. Additionally, RPMI8226 cells stably transfected to over-express either Bcl-2, Bcl-xL, or Mcl-1 are protected from cell death induced by CD86 or CD28 silencing. These data suggest that CD28 and CD86 are essential to prevent apoptosis of myeloma cells in vitro. To confirm these findings we determined the effects of CTLA4-Ig on myeloma survival. CTLA4-Ig inhibits CD86-CD28 signaling by binding to CD86, blocking its interaction with CD28. We found that treatment of RPMI8226 and MM1.s cells with CTLA4-Ig caused apoptosis in the myeloma cells after 2 days (23.9±3.9 for RPMI8226 and 20.4±6.2 for MM1.s, percent control apoptosis). Thus like normal plasma cells, CD28 and CD86 are required for the survival of myeloma cells. To determine why silencing of CD86 has a more potent effect than CD28 silencing on myeloma cell survival in 2 out of 3 cell lines, we investigated the effects of silencing on cell surface expression of each of these proteins. CD28 and CD86 mRNA and protein levels were silenced to similar levels by their cognate hairpins. However, in MM.1s and RPMI8226 we found that silencing of CD28 resulted in an increase in CD86 surface expression. This increase was also observed at the mRNA level and in the cells over-expressing Bcl-2 family members, indicating that this is not simply due to the selection of the highest expressing cells. These data suggest a feedback loop exists to regulate CD28-CD86 signaling in myeloma cells. Surprisingly, in the KMS18 cell line, we observe the converse effect, where silencing of CD86 resulted in upregulation of CD28. This provides a likely explanation for why these cells are less susceptible to CD86 silencing than the other two lines. Interestingly, blocking CD86 with CTLA4-Ig treatment also resulted in a modest upregulation in CD28 surface expression of MM.1s and RPMI8226, which suggests that silencing CD86 and binding of CD86 with a soluble receptor are not equivalent, and that multiple signaling feedback pathways exist to regulate the expression of this receptor-ligand pair that is necessary for myeloma cell survival. 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.

Sphingolipids as a novel target for the treatment of multiple myeloma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e19534-e19534
Author(s):  
Yubin Kang ◽  
Jagadish Kummetha Venketa
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Myeloma Cell ◽  
Sphingolipid Metabolism ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Novel Target

e19534 Background: Multiple myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ~10,600 deaths annually. MM remains an incurable disease and almost all patients will eventually relapse and become refractory to currently available therapeutic agents. There is an unmet need for better understanding the disease’s molecular pathways and for identifying novel therapeutic targets. Sphingolipid metabolism is being increasingly recognized as a key pathway in tumor cell proliferation and in tumor sensitivity to anticancer drugs. We hypothesize that altered sphingolipid metabolism plays an important role in the pathogenesis of MM, thus providing a novel target in the treatment of MM. Methods: We first assayed sphingolipid metabolism including sphingolipid metabolites and sphingolipid metabolizing genes in myeloma cell lines, in freshly isolated human primary CD138+myeloma cells, and in publically available dataset. We then tested the efficacy of the selective SK2 inhibitor (ABC294640) and the SK2 shRNA in killing myeloma cells in vitro. Results: 1) Compared to immortalized B cells, the levels of pro-apoptotic ceramides were decreased whereas the proliferative sphingosine 1-phosphate (S1P) was increased in myeloma cell lines. 2) The expression of several key sphingolipid-metabolizing genes including sphingosine kinase (SK) 1 and 2 was altered in freshly isolated human primary bone marrow myeloma cells and in publically available microarray dataset. 3) The selective SK2 inhibitor (ABC294640) induces apoptotic cell death and inhibits myeloma cell growth with an IC50of ~20 μM in 9 myeloma cell lines. 4) Interestingly, OPM-1 myeloma cell line was extremely sensitive to ABC294640 with an IC50of <5 µM whereas U266 myeloma cell line was resistant to ABC294640. SK2 shRNA induced apoptotic cell death in OPM-1, but not in U266 cells. We are currently investigating the molecular mechanisms underlying the resistance of U266 myeloma cells to ABC294640. Conclusions: Our data demonstrated that sphingolipid metabolism provides an attractive target in the treatment of refractory/relapased multiple myeloma.

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