Addiction to c-MYC in multiple myeloma

Blood ◽  
2012 ◽  
Vol 120 (12) ◽  
pp. 2450-2453 ◽  
Author(s):  
Toril Holien ◽  
Thea Kristin Våtsveen ◽  
Hanne Hella ◽  
Anders Waage ◽  
Anders Sundan

Abstract In multiple myeloma, c-MYC is activated and contributes to the malignant phenotype. Targeting MYC by short hairpin RNA induced cell death in myeloma cell lines; however, cell lines are generated from samples taken in advanced stages of the disease and may not reflect patient cells adequately. In this study, we used the selective small molecule inhibitor of MYC-MAX heterodimerization, 10058-F4, on myeloma cell lines as well as primary myeloma cells, and we show that inhibition of c-MYC activity efficiently induces myeloma cell death. Moreover, in cocultures of cell lines with bone marrow stromal cells from myeloma patients, the inhibitor still induces apoptosis. Our results provide further evidence that myeloma cells are addicted to c-MYC activity and that c-MYC is a promising therapeutic target in multiple myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3464-3464
Author(s):  
Shaji Kumar ◽  
Michael Timm ◽  
Michael P. Kline ◽  
Jessica L. Haug ◽  
Teresa K. Kimlinger ◽  
...  

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. The tumor suppressor gene p53, responsible for induction of cellular apoptosis in response to genotoxic stimuli, is relatively intact in most cases of myeloma. However, p53 mutations or deletion can occur late in the course of disease. Here we evaluate a novel small molecule inhibitor of the interaction between p53 and its negative regulator, MDM2, in the setting of myeloma. Methods and Results: Mi-63 was cytotoxic to several different myeloma cell lines with a median effect observed at approximately 2.5 μM in cell lines including MM1.S that express wild type p53 and between 10–15 μM in cells with mutated p53 as measured using an MTT cell viability assay. Additionally, Mi63 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. Mi63 was cytotoxic to myeloma cells under these conditions as well, at doses similar to those seen with myeloma cells alone. Mi63 was able to inhibit proliferation and induce apoptosis in myeloma cells in a dose- and time-dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. Treatment of myeloma cells with Mi63 was associated with early mitochondrial membrane depolarization, inversion of Bax/Bcl-2 ratio, and down regulation of Mcl-1, indicating induction of mitochondrial mechanisms of cell death. Mi63 was also cytotoxic to freshly isolated primary patient myeloma cells, inducing apoptosis in a dose-dependent manner. In the patient cells the drug appears to have a differential effect on the CD45 positive and negative cells. Conclusion: Mi-63 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. It clearly induces apoptosis in myeloma cells, with higher activity seen in cells with wild type p53. Given the lack of p53 abnormalities in most of the patients with myeloma, this drug alone or in combination is likely to have significant clinical activity. Studies combining this with various DNA damaging drugs are in progress. These studies will eventually form the framework for future clinical studies.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3005-3005
Author(s):  
Bjoern Jacobi ◽  
Lea Stroeher ◽  
Nadine Leuchtner ◽  
Hakim Echchannaoui ◽  
Alexander Desuki ◽  
...  

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.


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

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.


2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e19534-e19534
Author(s):  
Yubin Kang ◽  
Jagadish Kummetha Venketa

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.


Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1581-1581
Author(s):  
Shaji Kumar ◽  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
...  

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. Studies have shown elevated expression of anti-apoptotic proteins of the Bcl-2 family in MM cells, which appear to correlate with resistance to therapy with certain drugs. Hence, accelerating the apoptotic process by targeting the Bcl-2 family of proteins appears to be an attractive strategy for the treatment of MM. AT-101 is an orally bioavailable derivative of gossypol in cancer clinical trials, and is being developed by Ascenta Therapeutics. AT-101 behaves as a small molecule inhibitor of Bcl-2 and Bcl-XL, binding to the BH3-binding pocket of these proteins and inhibiting their ability to suppress the activity of pro-apoptotic proteins, resulting in apoptosis. Methods and Results: AT-101 was cytotoxic to several different myeloma cell lines with a median effect observed at around 5μM concentration using an MTT cell proliferation assay. Additionally, at similar doses AT-101 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. AT-101 was cytotoxic to myeloma cells under these conditions as well with a median effect at concentrations of 5–10μM. AT-101 was able to induce apoptosis in myeloma cells in a dose- and time dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. AT-101 also resulted in cytotoxicity of freshly isolated primary patient myeloma cells, inducing apoptosis in a dose dependent manner. We also studied the effect of AT-101 on levels of different pro- and anti-apoptotic proteins using flow cytometry on permeabilized cells. A time-dependent increase in the level of BAX was observed following treatment with AT-101 without any associated change in levels of Bcl-xL or Bcl-2. Further studies evaluating the combination of AT101 with other active myeloma agents as well as a detailed evaluation of its mechanisms in myeloma are ongoing. Conclusion: AT-101 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. More importantly, it has activity against cell lines resistant to conventional anti-myeloma agents. In addition, Phase I studies with this agent are currently ongoing in patients with solid tumors. The results from these studies form the rationale for early phase clinical trials in MM, either alone or in combination with other active therapies.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3163-3163 ◽  
Author(s):  
Jagadish Kummetha Venkata ◽  
Robert K Stuart ◽  
Luciano J Costa ◽  
Ningfei An ◽  
Houjian Cai ◽  
...  

Abstract Introduction 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 of the disease’s molecular pathways and identifying novel therapeutic targets. Sphingolipid metabolism is being increasingly recognized as a key pathway in cancer biology. In particular, sphingosine kinases (SK1 and SK2) provide a potential site for manipulation of the ceramide / sphingosine 1-phosphate (S1P) rheostat that regulates the balance between tumor cell proliferation and apoptosis, as well as tumor sensitivity to drugs. Currently, very little is known about sphingolipid metabolism in MM. We herein for the first time provide a detailed analysis of sphingolipid metabolism in MM and demonstrate the potential of targeting SK2 for the treatment of MM. Methods We first quantified sphingolipid metabolites and sphingolipid metabolizing genes in myeloma cell lines, in freshly isolated human primary CD138+ myeloma cells, and in a publically available gene expression dataset from MM patients. We then tested the anti-myeloma activity of SK2-specific shRNA and determined the efficacy of a selective SK2 inhibitor (ABC294640) in killing myeloma cell lines and primary human myeloma cells in vitro. The mechanistic pathway of apoptosis was analyzed by immunoblotting and flowcytometry. MM cell lines stably expressing luciferase and eGFP were generated for xenograft experiments and for in vitro co-cultures with stromal cells. Results From the publically available GSE6477 microarray data set, we found that one third of the genes involved in sphingolipid metabolism were significantly different in CD138+ MM cells from newly diagnosed MM patients compared to normal individuals, including SK2 and S1P receptors. In 5 MM cell lines compared to immortalized B cells (IBC), 19 key sphingolipid metabolites were measured, and we found that ceramides were significantly reduced whereas S1P was significantly increased. mRNA analyses of 11 sphingolipid metabolizing genes including S1P receptors in 7 MMs showed that SK1, SK2, and alkaline ceramidases were significantly increased compared to IBC. Furthermore, we isolated CD138+ myeloma cells from 21 MM patients and found that 13 of the patients had higher SK2 expression in CD138+ MM cells compared to CD138-cells. These data demonstrated abnormal sphingolipid metabolism and dys-regulated SK2 in myeloma cells. We generated SK2-specific shRNA and found that SK2 shRNA down-regulated SK2 mRNA, inhibited proliferation, and induced death in myeloma cells, suggesting that SK2 is important in myeloma cell survival. We then tested the efficacy of ABC294640 (the most-advanced, non-lipid SK2 inhibitor) in 6 MM cell lines. ABC294640 inhibited myeloma cell growth with an IC50s of ∼30 μM, including steroid-resistant and doxorubicin-resistant myeloma cells. ABC294640 inhibited MM cell growth as early as 6 hours after exposure and induced apoptotic cell death as demonstrated by Annexin V staining, PARP cleavage and caspase 9 activation. ABC294640 inhibited primary human CD138+MM cells with the same efficacy as with MM cell lines, demonstrating the potential of ABC294640 for the treatment of MM. Additionally, we found that blocking S1P receptors with FTY720 (a S1PR agonist with receptor degradation) induced apoptosis in MM cells. We performed extensive mechanistic and signaling pathway analyses and found that ABC294640 inhibited Mcl-1 and C-Myc expression, but had no effects on Bcl2. Furthermore, ABC294640 induced cell death by directing Mcl-1 to proteosomal degradation. MM is dependent on the bone marrow niche microenvironment for survival and progression. We found that ABC294640 was effective in inducing apoptosis in MM cells even in the presence of stromal cells. Finally, we are currently testing the in vivo effect of ABC294640 alone and in combination with bortezomib, thalidomide and dexamethasone in MM xenograft model transplanted with MM cells stably expressing luciferase. Our early preliminary results were encouraging. Conclusion Our data demonstrate that sphingolipid metabolism is abnormal and provides an attractive target in the treatment of refractory/relapsed MM. Disclosures: Costa: Otsuka: Research Funding.


Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5163-5163 ◽  
Author(s):  
Lawrence H. Boise ◽  
Alejo A. Morales ◽  
Delia Gutman ◽  
Kelvin P. Lee

Abstract Arsenic Trioxide (ATO) has been shown to be highly active against acute promyelocytic leukemia (APL) and has activity in several other diseases including multiple myeloma. While initial clinical trials in both APL and myeloma have suggested that melarsoprol results in greater dose-limiting toxicities than ATO, it is generally accepted that organic arsenicals are less toxic than inorganic arsenicals. Consequently, organic arsenicals that kill myeloma cells could be clinically more effective than ATO. Recently, several organic arsenicals were synthesized that have EC50s similar to ATO against cell line panels but are > 10-fold less toxic. One such compound, ZIO-101 is in phase I studies. Therefore we compared the ability of ZIO-101 and ATO to kill four myeloma cell lines that display differential sensitivity to ATO. The RPMI 8226 and U266 are less sensitive than the KMS11 and MM.1s lines. When dose response curves were generated comparing ATO and ZIO-101 at 24, 48 and 72 hrs we found that the U266, KMS11 and MM.1s lines were consistently 1–3 fold less sensitive to ZIO-101 than to ATO. However if one considers the number of atoms of elemental arsenic/molecule of drug, these data would suggest that the ability of these drugs to kill MM cell lines is similar. In contrast the 8226 line was more sensitive to ZIO-101. Additionally we have previously reported that ATO induces caspase-dependent and -independent cell death in a cell specific fashion in these lines. We found a similar pattern of caspase dependence with ZIO-101 where BocD-FMK, a caspase inhibitor, completely blocks ZIO-101-induced killing of U266, partially blocks killing of MM.1s and KMS11 and has no effect no killing of 8226. These data suggest that the downstream components of the death signaling pathway induced by ZIO-101 and ATO are similar. In contrast, initial responses to these drugs differ. We and others have reported that glutathione (GSH) is a critical regulator of ATO-induced cell death and have utilized ascorbic acid (AA) as a GSH depleting agent both in vitro as well as clinically. We therefore tested the effects of GSH depletion on ZIO-101 induced cell death in MM cell lines. Using concentrations of ATO and ZIO-101 that had similar activity, we determined the effects of both an inhibitor of GSH synthesis (BSO) as well as AA that can transient deplete GSH. BSO sensitized all 4 cell lines to both agents, however it was much more effective at sensitizing cells to ATO than to ZIO-101. Moreover while AA could sensitize cells to ATO, it actually protected cells from cell death induced by ZIO-101. Taken together these data suggest that ZIO-101 has activity against myeloma cells although factors that determine the potency of this compound are different than those for ATO. This may reflect differences in either metabolism or mechanism of action. Thus resistance to one form of arsenic does not preclude the use of another. A phase I/II study of ZIO-101 in myeloma is planned.


Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3446-3446 ◽  
Author(s):  
Ebenezer David ◽  
Rajni Sinha ◽  
Jonathan L. Kaufman ◽  
Sagar Lonial

Abstract Background: Perifosine is an oral AKT inhibitor which exerts a marked cytotoxic effect on human tumor cell lines. It is currently being tested in several phase II trials for the treatment of major cancers including multiple myeloma. While the proposed mechanism of action relates to downregulation of AKT expression, overepxression of constitutively active AKT does not abrogate perifosine induced cell death suggesting alternative mechanisms. Hypothesis: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2 ligand) effectively kills multiple myeloma cells in vitro after binding to their membrane specific receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). It is our hypothesis that DR4/DR5 upregulation occurs in response to perifosine treatment, and thus may be additive with exogenous TRAIL. Materials and Methods: TRAIL-sensitive myeloma cell lines (MM.1S, RPMI8226, MM.1R) and TRAIL- resistant myeloma cell lines (U266) were used in this study. Apoptosis was assessed by annexin-V binding assay using flow-cytometry and cell death was assessed by MTT assay. Recombinant human TRAIL, chimeras of DR4 and DR5 were obtained from R&D systems. Results: Perifosine alone(5μM and 10μM) induced apoptosis of MM.1S in 40% and 50% of the treated cells as measured by flow cytometry, that increased to 81% and 91% when 50ng/ml of TRAIL was added to 5μM and 10 μM of perifosine. TRAIL alone induced only nominal apoptosis. Use of the TRAIL resistant U266 cell line showed only minimal apoptosis in response to perifosine, TRAIL, or the combination of both agents. Perifosine also induced DR4 and DR5 expression in less than 2hrs upon the Perifosine exposure in MM.1S as shown by RT-PCR. The combination of perifosine and TRAIL was not sequence specific. Furthermore, we observed that the enhanced apoptosis induced by perifosine and TRAIL in combination was almost or partially blocked by the administration of the DR4 and DR5 blocking antibodies only in the case of MM.1S, MM.1R, RPMI8226 TRAIL sensitive cells lines. Apoptosis was completely blocked in the case of U266 TRAIL resistant cell line when the chimera antibodies were used with perifosine alone or in combination with TRAIL. Conclusion and future directions: Perifosine, an agent proposed to function via inhibition of p-AKT and PDK-1, may have other effects on cell cycle regulation and it pro-apoptotic effects may be partially related to the TRAIL pathway. Our data suggests that an additional mechanism of action relates to the effect perifosine has on DR4 and DR5 expression thus directly effecting apoptosis via the TRAIL mediated effects. The limited response the trail resistant cell line U266 cells suggest that the TRAIL resistant myeloma cells have less DR4 or DR5 surface receptors as compared to the TRAIL sensitive cell lines, MM.1S, MM.1R, and RPMI8226 further validating this alternative mechanism. Further experiments such as inhibition of DR4, DR5, and FADD by small interfering RNAs, RT-PCR, the response in primary myeloma cells and also using more TRAIL resistant cell lines to support our preliminary observations are currently in progress.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2720-2720
Author(s):  
Emmanouil Spanoudakis ◽  
Ming Hu ◽  
Kikkeri Naresh ◽  
Evangelos Terpos ◽  
Valeria Melo ◽  
...  

Abstract Downregulation of conventional HLA molecules from the surface of tumour cells is an important mechanism for tumour immune evasion, survival and progression. Whether CD1d, a non-conventional, glycolipid-presenting HLA class I-like molecule can affect tumour cell survival is not known. To test this we studied expression of surface CD1d on plasma cells from different stages of multiple myeloma (MM) using flow-cytometry. Expressing results as the ratio of the Geo MFI CD1d/isotype IgG1 we found that while CD1d expression was comparable between MGUS (n=8) and newly diagnosed MM patients (n=14; Geo MFI MGUS: 8.61±4.3 vs new MM: 7.1±4.72, p&gt;0.05), in relapsed/advanced disease CD1d was significantly lower (Geo MFI:1.92±0.9, p&lt;0.003 vs MGUS and new MM) and completely lost in 4 out of 5 myeloma cell lines at protein and RNA level. Further, 4 out of 8 paired, same-patient trephine biopsies stained with anti- CD1d showed drastic loss of CD1d expression in advanced compared to early disease. These results confirmed loss of CD1d expression during disease progression and suggested that CD1d impacts negatively on myeloma cell survival. Consistent with this, we found that engagement of CD1d by 2 different anti-CD1d mAbs and as compared to isotypic IgG or media control, induces cell death (i.e., Annexin+) of the CD1d-expressing B lymphoblastoid cell line C1R-CD1d, of myeloma cell lines with retrovirally restored expression of CD1d and purified, CD1d-expressing primary myeloma cells in a dose- and time-dependent manner, coincident with loss of mitochondrial membrane potential (MMP) as assessed by DioC3 staining. Biochemical analysis of relevant cell death pathways showed that MMP loss is associated with overexpression of the pro-apoptotic protein Bax but as demonstrated by immunoblotting and pharmacological inhibition it is caspase- independent. By introducing appropriate CD1d retroviral constructs into CD1d- myeloma cell lines we showed that anti-CD1d-induced cell death requires the cytoplasmic tail but not a Tyr residue critical for lysosomal sorting of CD1d. Finally, we found that anti-CD1d co-operates with anti-myeloma agents in the killing of myeloma cells. Thus, these findings provide evidence linking a novel function of CD1d in the regulation of cell death with tumour survival and progression and might have pathogenetic and therapeutic implications for other CD1d-expressing hematopoietic malignancies as well as myeloma.


2004 ◽  
Vol 52 (5) ◽  
pp. 335-344 ◽  
Author(s):  
Naomi Gronich ◽  
Liat Drucker ◽  
Hava Shapiro ◽  
Judith Radnay ◽  
Shai Yarkoni ◽  
...  

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.


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