Signaling Profile and Anti-Tumor Activity of the Novel HSP90 Inhibitor NVP-AUY922 in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4788-4788
Author(s):  
Thorsten Stühmer ◽  
Angela Zöllinger ◽  
Daniela Siegmund ◽  
Manik Chatterjee ◽  
Evelyn Grella ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Hsp90 Inhibitor ◽  
Dose Effect ◽  
Therapeutic Window ◽  
Large Set ◽  
Molecular Effects

Abstract We have recently shown that Hsp90 is overexpressed in multiple myeloma (MM) and critically contributes to tumor cell survival. Pharmacologic blockade of Hsp90 has consistently been shown to induce MM cell death. However, most data have been obtained with MM cell lines whereas knowledge about the molecular effects of pharmacologic Hsp90 blockade in primary tumor cells is lacking. Furthermore, these investigations have so far exclusively relied on geldanamycin derivatives. Here, we analyzed the anti-tumor effects of a novel diarylisoxazole-based Hsp90 inhibitor (NVP-AUY922) in a large set of primary MM samples and in MM cell lines. The majority of cell lines (n = 8), as well as most primary samples (n = 20), displayed profound apoptotic responses and steep dose-effect curves with EC50 values in the range of 5–15 nM and EC90 values between 8 and 25 nM. This effect was not attenuated in coculture with cells from the bone marrow microenvironment. Some cell lines and about a quarter of primary MM samples displayed greater resilience to drug treatment, with EC50 values but not EC90 values reached at concentrations up to 50 nM. Sensitivity of MM cells to the Hsp90 inhibitor was not correlated with TP53 mutation or Hsp70 induction levels. Western analyses of MM cell lines and flow cytometric analyses of antibody-stained Hsp90 client proteins in primary tumor cells showed that NVP-AUY922-treatment entailed molecular effects and pharmacodynamic properties consistent with abrogation of Hsp90 function. Consequently, downregulation of multiple signaling and survival pathways was detectable through, for example, decreases in the phosphorylated (activated) forms of extracellular signal-regulated kinase (ERK) 1 and 2, signal transducer and activator of transcription (STAT) 3 and glycogen synthase kinase-3beta. All samples treated displayed strong upregulation of Hsp70. Importantly, peripheral blood mononuclear cells as well as primary bone marrow stromal cells were much less affected by high (50–100 nM) concentrations of NVP-AUY922, showing that a therapeutic window might be established for the treatment of multiple myeloma. Taken together, NVP-AUY922 could be a promising new drug for the treatment of a majority of myeloma patients.

Effects of Integrin-Linked Kinase (ILK) Inhibitor QLT0267 on Multiple Myeloma Cells and Evaluation of ILK as a Therapeutic Target in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3529-3529
Author(s):  
Thorsten Stühmer ◽  
Torsten Steinbrunn ◽  
Evelyn Grella ◽  
Ralf C. Bargou
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Treatment Options ◽  
Therapeutic Window ◽  
Culture Dish ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells

Abstract Multiple myeloma (MM) is a fatal plasma cell tumor that accounts for about 1% of cancers. A hallmark of the disease is its location in the bone marrow where the tumor cells receive prosurvival support from the microenvironment and cause extensive osteolytic damage. Novel drugs are currently being developed into a range of new treatment options. However, because the problems of cancer relapse and eventual selection of therapy-resistant offspring remain, additional therapeutic targets should still be investigated. ILK is a multifunctional protein that, as an adaptor and/or as a kinase, may relay adhesion- and growth factor receptor-mediated signals to downstream signaling cascades that promote growth and survival. We have analysed the expression of ILK in MM cells and have tested the effects of a novel small molecule ILK-inhibitor (QLT0267; QLT Inc., Vancouver, Canada) in MM cell lines, primary MM tumor cells and healthy cells, respectively. ILK expression at either cDNA or protein level was detectable in virtually every MM sample tested. Treatment with QLT0267 for up to 3 days resulted in extensive apoptotic death in MM cell lines (EC50 values below 10 microM in 8/9 MM cell lines tested) and in a majority of primary (anti-CD138-purified) MM samples (EC50 values below 10 microM in 8/14 primary MM samples tested). Drug treatment led to rapid decreases in the levels of phospho-STAT3, phospho-GSK3beta and total Akt protein, whereas levels of ILK and of phospho-ERK were unaffected or, in the latter case, showed a slight increase. Similar to other current pharmacologic approaches, targeting ILK may have several detrimental impacts on the signaling network that sustains MM cells. Such pleiotropic effects could prove valuable for combination treatments. The survival of peripheral blood mononuclear cells and of bone marrow stromal cells (BMSCs) at 20 microM QLT0267 was just slightly affected, indicating that the scope for establishment of a therapeutic window in MM might exist. High (20 microM) concentrations of QLT0267 gradually (and reversibly) promoted detachment of BMSCs from the culture dish, indicating that the drug might be useful to temporarily impair their effectiveness to support myeloma cells. Taken together, these experiments provide a rationale to further explore the utility of ILK-inhibition for the treatment of MM.

Effect of the specific P53 stabilizer CBS9106 on multiple myeloma (MM)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 8601-8601
Author(s):  
H. Ikeda ◽  
T. Hideshima ◽  
G. Perrone ◽  
Y. Okawa ◽  
N. Raje ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Protein Expression ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
P53 Protein ◽  
Wild Type ◽  
Cycle Arrest ◽  
P53 Protein Expression

8601 Background: The mutations of P53 tumor suppressor protein are associated with progressive in Multiple Myeloma (MM), conversely, stabilization of P53 leads to cell cycle arrest and apoptosis. In this study, we examined p53 protein expression and demonstrated the effect of P53 stabilization using a novel specific P53 stabilizer CBS9106 in MM. Method: We examined P53 protein expression using Immunoblot analysis, as well as the growth inhibitory effect of CBS9106 in MM cell lines and primary tumor cells from MM patients. We also defined whether CBS9106 can overcome the growth promoting effect of exogenous cytokines and bone marrow stroma cells (BMSCs) using [3H]-thymidine uptake assay. Results: Expression of P53 protein was observed in 3/3 primary tumor cells from MM patients and 6/6 MM cell lines. CBS9106 at low nM levels triggered cytotoxicity against p53 wild type MM cell lines and primary tumor cells from MM patients, associated with phosphorylation of P53 (serine15 and 20). In contrast, CBS9106 did not affect the survival of normal peripheral blood mononuclear cells from healthy volunteers at concentrations as high as 10 μM. This agent also induced G1 cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, -8, -9 and PARP. Neither growth stimulating cytokines (IL-6 and IGF-1) nor BMSCs protected against apoptotic effect of CBS9106. Moreover, we demonstrate that combination of CBS9106 with MDM2 inhibitor Nutrin3 or proteasome inhibitor bortezomib induces synergistic anti-MM activity in both P53 wild type MM cell lines and primary tumor cells from MM patients. Conclusions: Stabilizing P53 by CBS9106 represents a novel promising p53-based therapy in MM. These results provide the preclinical framework supporting evaluation of CBS9106 in clinical trials to improve patient outcome in MM. No significant financial relationships to disclose.

Stromal Cell IL-8 Production Is Increased in MM and May Impact Both Tumor Cells and the Microenvironment.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2508-2508
Author(s):  
Michael Kline ◽  
Kathleen Donovan ◽  
Linda Wellik ◽  
Christopher Lust ◽  
Wendy Jin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Microvessel Density ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Clone ◽  
Angiogenic Factor ◽  
Antibody Array

Abstract Background: Multiple myeloma (MM) is an incurable hematological malignancy characterized by the expansion of a plasma cell clone that localizes to the bone marrow. Stromal cells residing in the bone marrow respond to signals from MM cells and other cell types by producing cytokines and other proteins that stimulate tumor cell growth, survival, adhesion, migration, and drug resistance. We have examined the proteins produced by stromal cells in response to stimulation by bone marrow from patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and MM. Furthermore, we have begun analyzing the properties of one of these proteins, the pro-angiogenic chemokine IL-8, in MM. Methods: Bone marrow aspirates from patients with MGUS (n=3), SMM (n=7), and MM (n=6) were cultured for 48 hours, and the culture supernatants were incubated with stromal cells for an additional 48 hours. Protein levels were analyzed using antibody array and ELISA. Microvessel density (MVD) was determined as a measure of angiogenesis in patient bone marrow samples using CD34 staining. Flow cytometry analysis of MM cell lines and patient bone marrow samples was performed using monoclonal antibodies against IL-8 receptors CXCR1 and CXCR2. Results and Conclusion: We observed a significant increase in stromal cell IL-8 production stimulated by bone marrow cells from patients with active myeloma and a subset of SMM patients (16.67 ± 9.82 ng/ml) in comparison to bone marrow of patients with MGUS and all other SMM patients (0.55 ± 0.17 ng/ml; P=0.0004). Use of an IL-1 inhibitor and recombinant IL-1β demonstrated that IL-8 production was dependent upon IL-1β signaling. Increased BM microvessel density correlated with stimulation of stromal cell IL-8 production (P=0.0005). Furthermore, the majority of MM cell lines (7/9) and MM patient plasma cells were found to express IL-8 receptors CXCR1 and CXCR2. In addition to its function as a pro-angiogenic factor, IL-8 may directly influence MM cells through its CXCR1 and CXCR2 receptors. We conclude that stromal cell IL-8 production parallels MM disease activity, is IL-1β induced, correlates with bone marrow angiogenesis, and may influence MM disease via impact upon both the microenvironment and tumor cells.

Patulin Acts Synergistically with Doxorubicin and Bortezomib in Cytotoxicity against Hematological Malignancies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2664-2664
Author(s):  
Alex Hessel ◽  
Malefa Tselanyane ◽  
Fengrong Wang ◽  
Ebenezer David ◽  
Sagar Lonial ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Primary Tumor ◽  
Dose Effect ◽  
Tumor Cell Lines ◽  
Synergism And Antagonism

Abstract Patulin (4-Hydroxy-4H-furo 3,2-C-pyran-2(6H)-one) is a first-in-class mycotoxin under development as a novel chemotherapeutic agent. The mechanism of action of Patulin has been reported to include activation of mitogen activated protein kinases (MAPKs) and generation of reactive oxygen species (ROS). We have previously shown Patulin to have activity against myeloma, leukemia, and lymphoma cell lines, as well as primary tumor cells in clinical samples from patients with these diagnoses (Wang BLOOD 2007). Moreover, we have shown that Patulin specifically and potently targets tumor cells over normal cells and effectively killed primary tumor cells of patients with refractory illness. The aim of this study was to determine whether Patulin acts synergistically with the topoisomerase inhibitor doxorubicin or the proteasome inhibitor bortezomib. We hypothesize that drugs with non-cross-reactive modes of action could be complementary. Human tumor cell lines from B-cell lymphoma (DAUDI), T-Cell leukemia and lymphoma (Jurkat and H9, respectively), and myeloid leukemia (HL60) malignancies were tested for their sensitivity to single agents Patulin, bortezomib, and doxorubicin as well as Patulin in combination with the latter two agents (Patulin and bortezomib, P + B; Patulin and doxorubicin, P + D). Cells were treated with a range of concentrations of each single agent and the drugs in combination over 24 hours. Following treatment, cell metabolic activity was assessed using a microculture tetrazolium (MTT) assay and cell viability was assessed by flow cytometry using Annexin V and propidium iodide (PI) staining. Dose-effect curves, median effect plots, and combination index (CI) values were generated in the Compusyn software program for each target cell population. Median-effect doses (IC50s) of individual drugs were interpolated using the y-intercept of median-effect plots. Three dose-effect data points were used to create a range of CI values at different fractions of affect (fa). The lower and upper values of the CI range were used to characterize drug combinations as synergistic, antagonistic, or additive based on Chou’s Symbols for Synergism and Antagonism using CI analysis (Table 1). The role for ROS in the mechanism of action of Patulin was confirmed by flow cytometry showing increased levels of ROS in cell lines following Patulin exposure. Preincubation of cell lines with N-acetyl cysteine (NAC) or concurrent exposure to Patulin and NAC abrogated the cytotoxic activity of the mycotoxin. H9 cells were most sensitive to the effects of Patulin, with an IC50 of 1.2 μM. Combinations of P + B acted synergistically against Jurkat, H9, DAUDI, and HL60 tumor cells; however, P + B also demonstrated moderate antagonism against the Jurkat and H9 cell lines (Table 2). Likewise, combinations of P + D interacted synergistically against Jurkat, H9, DAUDI, and HL60 tumor cell lines while simultaneously demonstrating strong antagonism against the H9 cell line. Patulin kills leukemia and lymphoma cells via generation of intracellular ROS. Synergy of Patulin with either bortezomib or doxorubicin in leukemia and lymphoma cell lines indicates a distinct mechanism of action for the mycotoxin and compared to other chemotherapeutics and supports the rationale for continued development of Patulin as a novel chemotherapeutic mycotoxin. TABLE 1. Chou’s Symbols for Synergism and Antagonism using CI Analysis CI Description < 0.1 Very Strong Synergism 0.1–0.3 Strong Synergism 0.3–0.7 Synergism 0.7–0.85 Moderate Synergism 0.85–0.90 Slight Synergism 0.90–1.10 Nearly Additive 1.10–1.20 Slight Antagonism 1.20–1.45 Moderate Antagonism 1.45–3.3 Antagonism 3.3–10 Strong Antagonism > 10 Very Strong Antagonism TABLE 2. IC50 of single agents patulin, bortezomib, and doxorubicin and CI in hematological cancer cell lines Tumor cell line IC50 of patulin (μM) IC50 of bortezomib (nM) IC50 of doxorubicin (μM) CI: P + B CI: P + D T-Cell Jurkat 1.16 5600 5.2 0.6 -- 1.2 0.1 -- 1.2 H9 1.2 14 0.33 0.4 -- > 1 0.3 -- > 1 B-Cell DAUDI 0.98 1.1 0.19 < 0.1 -- 0.1 < 0.1 -- 0.3 Myeloid HL60 1.7 0.42 0.07 < 0.1 -- 0.1 < 0.1

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

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

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

The Overexpression of Inactive Phosphorylated Raf-1 Kinase Inhibitory Protein (RKIP) in Multiple Myeloma (MM) Regulates the Resistance to Bortezomib-Induced Cytotoxicity: Reversal of Resistance by the PKC Inhibitor Bisindolylmalemide

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2892-2892
Author(s):  
Sara Huerta-Yepez ◽  
Stavroula Baritaki ◽  
Hong Liu ◽  
Karla MC Mendez-Maldonado ◽  
Manuel L Penichet ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Western Blot ◽  
Tumor Cells ◽  
Clinical Significance ◽  
Cell Lines ◽  
Cytotoxic Drugs ◽  
Inhibitory Protein ◽  
Pkc Inhibitor ◽  
Phosphorylated Form

Abstract Abstract 2892 Multiple myeloma (MM) is an incurable hematological malignancy. Thalidomide, bortezomib and lenalidomide have emerged as highly active agents in the treatment of MM. Although initial clinical responses to drug therapy are achieved, a significant number of MM patients relapses and no longer responds to further treatments. Hence, the mechanism of resistance of MM to current therapeutic regimens remains elusive. It will be useful to identify prognostic markers that can predict the poor response of patients to therapies and to develop novel therapeutics for these patients. Our studies and those of others have recently identified a gene product, Raf-1 kinase inhibitory protein (RKIP) that has been shown to be involved in the regulation of tumor sensitivity to drugs and that has been found to be poorly expressed in different histological resistant tumors studied. RKIP is a member of the phosphatidylethanolamine-binding protein (PEBP) that functions as a potent inhibitor of different kinases of the Raf/MAPK (Raf-1 kinase) and NF-k B (TAK-1, NIK) activation pathways, and, thereby, antagonizing both cell survival and the expression of anti-apoptotic gene products. RKIP activity undergoes a post translational modification involving PKC–meditated phosphorylation at Ser 153 that inhibits its association with RAF/MAPK and NF-k B. The objective of this study was to examine the expression and function of RKIP and phospho-RKIP (p-RKIP) in MM and examined their clinical significance. MM cell lines and bone marrow-derived MM tissues were analyzed by both immunohistochemistry (IHC) and western blot for the expression of RKIP and p-RKIP. The antibody to RKIP recognizes both non-phospho and p-RKIP whereas the antibody against p-RKIP is specific. We demonstrate the unusual overexpression of RKIP in MM cell lines and tissues and the majority was in its inactive phosphorylated form. The expression of both forms in normal bone marrow was very low. Analysis of the ratio of RKIP/p-RKIP suggested its clinical significance wereby a higher ratio correlated with poor progression and a low ratio correlated with progression. The overexpression of p-RKIP suggested that it maintains the survival and the resistance of MM to cytotoxic drugs. We hypothesized that the dephosphorylation of RKIP may render RKIP active and modify the resistance to cytotoxic drugs. This hypothesis was tested in MM cell lines. Tumor cells were treated with the PKC inhibitor bisindolylmalemide (BIM) (5–10 μg/ml) for different times and complete inhibition of p-RKIP as detected as early as 4 h, while non phospho-RKIP was preserved, as detected by IHC and western blot. Treatment with BIM and followed with bortezomib (2.5-nM) resulted in significant cytotoxicity of the bortezomib-resistant MM cells. Overall, the findings demonstrate the unusual overexpression of p-RKIP in MM cells that plays an important role in survival and maintenance of tumor cell-resistance to cytotoxic drugs. The reversal of the active non-phosphorylated form of RKIP by the use of the specific PKC inhibitor rendered the tumor cells sensitive to bortezomib-induced cytotoxicity. These findings support the clinical application of specific PKC inhibitors in combination with bortezomib or other drugs in the treatment of patients who are resistant to conventional therapies. Disclosures: No relevant conflicts of interest to declare.

Centrosomal Clustering – a Novel Therapeutic Target for Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 300-300 ◽  
Author(s):  
Marc S Raab ◽  
Iris Breitkreutz ◽  
Blanka Rebacz ◽  
Thomas O Larsen ◽  
Ludmila Wagner ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Growth Inhibition ◽  
Tumor Cells ◽  
Cell Lines ◽  
Small Molecule ◽  
Therapeutic Target ◽  
Malignant Cells ◽  
Multipolar Spindles

Abstract Abstract 300 The lack of tumor-specific targets that would allow for the selective eradication of malignant cells without affecting healthy tissues is a major challenge in the development of novel therapies for multiple myeloma (MM). In contrast to normal cells, malignant plasma cells frequently contain multiple centrosomes, associated with the transient formation of multipolar mitotic spindles that lead to segregation defects and chromosomal instability. As in most tumor types, mitotic stability in these cells is maintained by coalescence of multiple centrosomes into two functional spindle poles, termed “centrosomal clustering”. As we have recently shown, this mechanism is an attractive therapeutic target with specificity for tumor cells. To identify potent and selective inhibitors of centrosomal clustering, we performed a phenotype-based small molecule screen in order to force tumor cells with supernumerary centrosomes to undergo multipolar mitoses resulting in apoptotic cell death. We here describe the characterization of a novel small molecule GF-15, a derivative of griseofulvin, as a potent inhibitor of centrosomal clustering, thereby inducing multipolar spindles followed by apoptosis in MM cells. We tested a wide array of MM cell lines, including those resistant to conventional chemotherapeutic agents, and primary patient cells. We found mean inhibitory concentrations (IC50) of proliferation and survival in the range of 1-5 mM, associated with annexin V conversion and activation of caspases 8, 9, and 3. Importantly, GF-15 also overcomes the tumor cell growth advantage conferred by both bone marrow stromal cell-MM, and endothelial cell-MM, co-culture systems. Moreover, non-malignant cells without supernumerary centrosomes like activated PBMCs, immortalized hepatocytes, and bone marrow stromal cells did not reach their IC50 at doses of up to 50 mM. To further demonstrate the specificity of GF-15, we generated resistant MM cell lines by long-term culture with sub-IC50 doses of GF-15. In resistant cell lines, therapeutic doses of GF-15 no longer induce multipolar spindles, consistent with a significant loss of centrosomal aberrations in these cells, as observed by immunoflourescence microscopy. Mechanistically, cell cycle analysis of synchronized MM cells showed marked G2/M arrest within 12-16h followed by a dramatic increase of the sub-G1 fraction after treatment with GF-15. In addition, short term treatment with GF-15 was associated with inhibition of VEGF- and IGF1-triggered MM cell migration. Co-treatment assays to assess potential partners for therapeutic combinations revealed at least additive effects for GF-15 together with bortezomib and marked synergism with paclitaxel at very low doses (1-5 nM), while the combination with melphalan resulted in antagonistic effects due to the S-phase arrest of tumor cells induced by melphalan. Finally and most importantly, i.p. as well as oral treatment of murine xenograft models of human MM resulted in tumor growth inhibition and significantly prolonged survival in vivo. Growth inhibition of xenograft tumor samples was associated with a dramatic increase of mitotic aberrations and multipolarity, as assessed by immunohistochemistry. In vivo biodistribution studies are ongoing. Taken together, our results demonstrate the in vitro and in vivo anti-tumor efficacy of a prototype small molecule inhibitor of centrosomal clustering with specificity for tumor cells, and therefore strongly support its further evaluation and development as a lead compound of a new class of therapeutics for human malignancies. Disclosures: No relevant conflicts of interest to declare.

The mTOR Inhibitor RAD001 (everolimus) Is Active Against Multiple Myeloma Cells In Vitro and in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1496-1496 ◽  
Author(s):  
Nicholas Mitsiades ◽  
Ciaran McMullan ◽  
Vassiliki Poulaki ◽  
Joseph Negri ◽  
Noopur Raje ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Mtor Inhibitor ◽  
Alkylating Agents ◽  
Anti Tumor Activity

Abstract We have recently shown that tumor cell proliferation, survival and drug-resistance in multiple myeloma (MM) and a broad range of other tumors is critically influenced by insulin-like growth factors (IGFs) and their receptor (IGF-1R) (Cancer Cell2004;5:221–30). Among the pleiotropic signaling cascades downstream of IGF-1R activation, we focused on the functional implications and therapeutic targeting of the Akt/p70S6K/mTOR axis, particularly of mTOR (mammalian Target of Rapamycin), due to its regulatory role on cellular bioenergetics, a key aspect of tumor pathophysiology. Herein, we describe the in vitro and in vivo profiles of anti-tumor activity of the selective mTOR inhibitor RAD001 (Everolimus, Novartis AG). Using in vitro MTT assays, we observed that RAD001 is active (at nM concentrations) against a broad range of tumor cells, including >40 MM cell lines and >10 primary MM tumor cells (including cell lines or primary cells resistant to Dex, alkylating agents, anthracyclines, thalidomide (Thal), immunomodulatory Thal derivatives, bortezomib, and/or Apo2L/TRAIL), without significant impact on viability of normal hematopoietic cells or other normal tissues (e.g. bone marrow stromal cells), and its anti-MM effect was not blocked by forced overexpression of Bcl-2 or constitutively active Akt. While cytokine- or cell adhesion-mediated interactions with the bone marrow (BM) microenvironment (e.g. BM stromal cells) protects MM cells from conventional therapies (e.g. Dex or cytotoxic chemotherapy), RAD001 was able to overcome this protective effect in co-culture models of MM cells with BM stromal cells or in vitro MM cell exposure to survival factors, e.g. IL-6 or IGF-I. Furthermore, RAD001 sensitized MM cells to other anti-MM therapeutics, e.g. dexamethasone, cytotoxic chemotherapeutics, or the proteasome inhibitor bortezomib, even in cases of primary MM tumor cells refractory to these respective agents. Using hierarchical clustering analyses and relevance network algorithms, we found that the pattern of MM cell dose-response relationships to RAD001 is clearly distinct from the patterns of sensitivity or resistance to other conventional or investigational anti-MM drugs. This further supports the notion that RAD001 confers a constellation of pro-apoptotic/anti-proliferative molecular sequelae distinct from those of currently available anti-MM drugs, and also suggests that RAD001 may have anti-tumor activity even against subgroups of MM which may be resistant to other novel therapies which that are currently in clinical development. Importantly, administration of RAD001 in a SCID/NOD mice model of diffuse MM bone had in vivo anti-tumor activity, including suppression of MM tumor burden and prolongation of survival (p<0.01, log-rank test). These studies highlight an important role for mTOR in growth/survival of human MM cells and provide proof-of-principle for future clinical studies of mTOR inhibitors for the treatment of MM and other plasma cell dyscrasias.

scholarly journals Metastatic Esophageal Carcinoma Cells Exhibit Reduced Adhesion Strength and Enhanced Thermogenesis

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1213
Author(s):  
Zihe Huo ◽  
Mariana Sá Santos ◽  
Astrid Drenckhan ◽  
Stefan Holland-Cunz ◽  
Jakob R. Izbicki ◽  
...  
Keyword(s):  
Esophageal Carcinoma ◽  
Tumor Cells ◽  
Cell Lines ◽  
Adhesion Strength ◽  
Primary Tumor ◽  
Carcinoma Cell ◽  
Metastatic Potential ◽  
Primary Tumors ◽  
Metastatic Cell ◽  
Carcinoma Cell Lines

Despite continuous improvements in multimodal therapeutic strategies, esophageal carcinoma maintains a high mortality rate. Metastases are a major life-limiting component; however, very little is known about why some tumors have high metastatic potential and others not. In this study, we investigated thermogenic activity and adhesion strength of primary tumor cells and corresponding metastatic cell lines derived from two patients with metastatic adenocarcinoma of the esophagus. We hypothesized that the increased metastatic potential of the metastatic cell lines correlates with higher thermogenic activity and decreased adhesion strength. Our data show that patient-derived metastatic esophageal tumor cells have a higher thermogenic profile as well as a decreased adhesion strength compared to their corresponding primary tumor cells. Using two paired esophageal carcinoma cell lines of primary tumor and lymph nodes makes the data unique. Both higher specific thermogenesis profile and decreased adhesion strength are associated with a higher metastatic potential. They are in congruence with the clinical patient presentation. Understanding these functional, biophysical properties of patient derived esophageal carcinoma cell lines will enable us to gain further insight into the mechanisms of metastatic potential of primary tumors and metastases. Microcalorimetric evaluation will furthermore allow for rapid assessment of new treatment options for primary tumor and metastases aimed at decreasing the metastatic potential.

scholarly journals Establishment of Cell Lines from Both Myeloma Bone Marrow and Plasmacytoma: SNU_MM1393_BM and SNU_MM1393_SC from a Single Patient

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Youngil Koh ◽  
Woo-June Jung ◽  
Kwang-Sung Ahn ◽  
Sung-Soo Yoon
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Clonal Evolution ◽  
Myeloma Cell ◽  
Mouse Bone Marrow ◽  
Single Patient ◽  
U266 Cell

Purpose.We tried to establish clinically relevant human myeloma cell lines that can contribute to the understanding of multiple myeloma (MM).Materials and Methods.Mononuclear cells obtained from MM patient’s bone marrow were injected via tail vein in an NRG/SCID mouse. Fourteen weeks after the injection, tumor developed at subcutis of the mouse. The engraftment of MM cells into mouse bone marrow (BM) was also observed. We separated and cultured cells from subcutis and BM.Results.After the separation and culture of cells from subcutis and BM, we established two cell lines originating from a single patient (SNU_MM1393_BM and SNU_MM1393_SC). Karyotype of the two newly established MM cell lines showed tetraploidy which is different from the karyotype of the patient (diploidy) indicating clonal evolution. In contrast to SNU_MM1393_BM, cell proliferation of SNU_MM1393_SC was IL-6 independent. SNU_MM1393_BM and SNU_MM1393_SC showed high degree of resistance against bortezomib compared to U266 cell line. SNU_MM1393_BM had the greater lethality compared to SNU_MM1393_SC.Conclusion.Two cell lines harboring different site tropisms established from a single patient showed differences in cytokine response and lethality. Our newly established cell lines could be used as a tool to understand the biology of multiple myeloma.

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