Significant in-Vitro Activity of a Novel JAK2 Inhibitor in Multiple Myeloma Associated with Preferential Cytotoxicity for CD45+ Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2848-2848
Author(s):  
Vijay Ramakrishnan ◽  
Jessica Haug ◽  
Teresa Kimlinger ◽  
Timothy Halling ◽  
Linda Wellik ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Research Funding ◽  
Myeloma Cell ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Myeloma Cells ◽  
Stat Pathway

Abstract Abstract 2848 Poster Board II-824 Background: Multiple myeloma remains incurable with current therapies and novel approaches based on disease biology are needed. IL-6 is a critical cytokine involved in myeloma cell proliferation and survival and exerts its activity primarily through the JAK/STAT pathway. In addition to IL6, other cytokines are also believed to cross talk with the JAK/STAT pathway, making it a crucial interface for survival signals. It has been implicated in myeloma cell interaction with the microenvironment and resistance to apoptotic stimuli from different drugs, and represents a potential therapeutic target. We examined the pre-clinical activity of a novel JAK2 tyrosine kinase inhibitor TG101209. Methods: TG101209 (N-tert-butyl-3-(5-methyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-ylamino)-benzenesulfonamide) was synthesized by TargeGen Inc. (San Diego, CA, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and using Apo2.7 in primary patient cells. CD45 expression was estimated using flow cytometry and cells were gated by their CD45 expression to assess differential effects of the drug. Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results: TG101209 resulted in a dose and time dependent inhibition of cell growth in the MM cell lines tested. Most of the cytotoxicity was evident by 48 hours, with minimal increase seen up to 96 hours of incubation. At 48 hours of incubation, the median inhibitory concentration was between 2 and 4uM with similar IC50 seen for myeloma cell lines sensitive or resistant to conventional therapies. The IC50s were maintained when the cells were treated in co-culture with stromal cells or in the presence of IL6, IGF or VEGF. Increasing doses of IL6 was not able to rescue the cells from the drug. Dose dependent decrease in proliferation of the cell lines was evidenced by decreased thymidine incorporation. Apoptotic changes in cells following drug treatment was confirmed by flow cytometry for Annexin and PI. Cleavage of caspases 3, 8 and 9 were confirmed on flow cytometry. Addition of the pan-caspase inhibitor zvad-fmk did not prevent drug-induced apoptosis confirming non-caspase mediated mechanisms of cell death as well. Primary myeloma cells from several patients were treated with increasing doses of the drug and IC50 similar to cell lines were seen in 8/10 patient samples tested. Interestingly, evaluation of U266 cell lines, which have a mix of CD45+ and negative cells as well as primary patient cells demonstrated more profound cytotoxicity and anti-proliferative activity of the drug on the CD45+ population relative to the CD45- cells. Immunoblotting studies demonstrated significant down regulation of IL-6 induced pSTAT3 with minor effects on the pERK and pAkt. The effect on pSAT3 was sustained compared to that on pERK and pAkt. This was accompanied by significant down regulation of Bcl-xL. Studies in a mouse model of myeloma are planned. Conclusion: These studies demonstrate significant in-vitro activity of JAK2 inhibition in multiple myeloma. In particular, the preferential targeting of CD45 cells, considered to reflect the proliferative compartment in myeloma holds out the promise for more sustained impact on the disease from a therapeutic standpoint. This is likely explained by the increased sensitivity of the CD45 cells to cytokines as a result of higher expression of different cytokine receptors as has been previously shown. This leads to increased activity of and dependence of the cells on the JAK-STAT pathway and likely explains the increased effect of the pathway inhibition. These studies form the framework for clinical evaluation of the drug in the setting of myeloma. Disclosures: Kumar: CELGENE: Research Funding; MILLENNIUM: Research Funding; BAYER: Research Funding; GENZYME: Research Funding; NOVARTIS: Research Funding.

Thymoglobulin (Polyclonal Rabbit Anti Thymocyte Globulin) Has In Vitro Activity in Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5190-5190
Author(s):  
Shaji Kumar ◽  
Michael Timm ◽  
Terry Kimlinger ◽  
Michael Kline ◽  
Jessica L. Haug ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Myeloma Cells ◽  
Polyclonal Rabbit

Abstract Background: Multiple myeloma is a plasma cell malignancy that remains incurable with current approaches and newer therapies are needed to improve the outcome of patients with MM. While monoclonal antibody base therapies have been successful in some of the hematological malignancies, especially lymphoma, such approaches have not been very useful in the setting of myeloma. Targeting of antigens like CD138 on the myeloma cell surface has been hampered by the ubiquitous nature of this protein in the body. Thymoglobulin (polyclonal rabbit antithymocyte globulin, Genzyme) has been extensively evaluated in the setting of allogeneic blood and marrow transplantation and is currently in clinical use. Given the polyclonal nature of this product, it has antibodies against several B cell antigens and forms the rationale for its evaluation in B cell malignancies like myeloma. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity following drug treatment was measured using the MTT viability assay. Drug induced apoptosis in the cell lines was measured by flow cytometry after staining with Annexin V-FITC and propidium iodide (PI). Apoptosis in primary patient derived plasma cells following treatment was measured after staining for Apo 2.7. Results: rATG was cytotoxic in vitro to several MM cell lines (RPMI 8226, U266, OPM1, OPM2) including the IL-6 dependent cell line Kas6/1. The LC50 in most of the cytotoxicity assays was around 1 mg/mL. Additionally, rATG was cytotoxic MM cell lines resistant to conventional agents such as doxorubicin (Dox40), melphalan (LR5) and dexamethasone (MM1R). The drug retained its cytotoxicity when myeloma cells were grown in the presence of various cytokines like IL-6, IGF-1 and VEGF. rATG treatment resulted in a time and dose dependent induction of apoptosis in MM cell lines. rATG was also able to induce apoptosis of freshly isolated myeloma cells from patient marrows. When tested in combination with other anti-myeloma agents an additive effect was seen with doxorubicin, PS341 and melphalan. Conclusions: Thymoglobulin appear to have in vitro activity against various myeloma cell lines as well as patient derived primary myeloma cells. Ability of the drug to overcome resistance to conventional drugs as well as the effect of combining rATG with these agents points towards non-overlapping mechanisms of action. Ongoing studies are trying to identify the particular B-cell antigens that are targeted by correlating response to expression of various B cell antigens expressed by these cell lines. These studies will provide the rational for future clinical development of this agent in myeloma alone or in combination with other agents.

AT-101, a Small Molecule Inhibitor of Bcl-2 Family Proteins, Has Significant In Vitro Activity in Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1581-1581
Author(s):  
Shaji Kumar ◽  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Clinical Trials ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Time Dependent ◽  
Myeloma Cells ◽  
Molecule Inhibitor ◽  
Apoptotic Proteins

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.

Significant In Vitro Activity of a Novel Inhibitor of Apoptosis (IAP) Inhibitor LC161 In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2998-2998
Author(s):  
Jessica Haug ◽  
Vijay G. Ramakrishnan ◽  
Teresa Kimlinger ◽  
Timothy Halling ◽  
Linda Wellik ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Research Funding ◽  
In Vitro Activity ◽  
Disease Biology ◽  
Marker Proteins ◽  
Dose Dependent

Abstract Abstract 2998 Background: Multiple myeloma remains incurable with current therapies and novel approaches based on disease biology are needed. Inhibitors of apoptosis (IAP) proteins represent a conserved group of proteins that are important regulators of apoptosis. X-linked IAP (XIAP) is the best studied IAP and inhibits pro-apoptotic caspases 3, 7 and 9. Multiple myeloma (MM) cell lines express high levels of XIAP and levels are further increased when stimulated by cytokines IL6 and IGF-1, both secreted in copious amounts in the myeloma microenvironment. IL6 and IGF1 up-regulate XIAP by activating the NF-κB, MAPK and PI3K signaling pathways that are commonly aberrant in MM and other tumors. XIAP mRNA contains an internal ribosomal entry site (IRES) sequence in the 5` untranslated region. IRES sequences enable direct ribosome recruitment and aid in translation that is independent of cap-mediated translation. Thus, molecules like mTOR inhibitors might not lead to XIAP downregulation. This was observed when rapamycin, an mTOR inhibitor, was used on MM cell lines. XIAP protein levels were not reduced due to the IRES sequence which leads to translation that is independent of the 5` cap and 4EBP1. Thus XIAP inhibitors might be able to overcome resistance associated with rapamycin treatment. Methods: LC161 was synthesized by Novartis Inc. (Basel, Switzerland). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results: LC161 treatment resulted in a dose and time dependent inhibition of cell growth in the MM cell lines tested. Most of the cytotoxicity was evident by 72 hours, with minimal increase seen up to 96 hours of incubation. At 72 hours of incubation, the median inhibitory concentration varied considerably between various cell lines with an IC50 range of 2.5–25μ M. The IC50s were maintained when the cells were treated in co-culture with stromal cells or in the presence of IL6, IGF or VEGF. Dose-dependent decrease in proliferation of the cell lines was evidenced by decreased thymidine incorporation. Apoptotic changes in cells following drug treatment was confirmed by flow cytometry for Annexin and PI. Cleavage of caspases 3, 8 and 9 were confirmed on flow cytometry. Primary myeloma cells from patients were treated with increasing doses of the drug and dose dependent increase in apoptosis was observed. Immunoblotting studies demonstrated dose dependent significant down regulation of Xiap, cIAP1, cIAP2 and surviving and up-regulation of activated caspases 3, 8 and 9 and PARP. Furthermore, LC161 resulted in down regulation of pAkt, canonical and non-canonical NF-κB, pJNK, p-p38MAPK, c-Myc, Bcl-xL and Mcl1 and up-regulation of pErk and Bcl-2. We are currently examining basal levels of expression of the IAP proteins (Xiap, cIAP1 and cIAP2), pAkt and pErk in various MM cell lines to identify marker proteins that might predict response to this class of drug. In addition, our initial studies of LC161 in combination with the proteasome inhibitor bortezomib demonstrated synergy in killing MM cells in vitro. Additional combinations including inhibitors of the Akt/mTOR pathway and MEK/Erk pathway are currently been done. Conclusion: These studies demonstrate significant in vitro activity of LC161 in MM. Our results suggest the presence of two populations one very sensitive to IAP inhibition and one relatively less sensitive. Our current studies will help identify marker proteins that might predict response to LC161 treatment. We are currently testing LC261 in combination with known inhibitors of the other important signaling pathways implicated in MM disease biology as well as in-vivo experiments in mouse models. Performing these experiments will further validate the efficacy of LC161 as an anti-MM agent and form the basis for it to be taken up for clinical evaluation either as a single agent or in combination with other agent(s). Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

In vitro activity of dimethylarsinic acid against human leukemia and multiple myeloma cell lines

2003 ◽  
Vol 51 (5) ◽  
pp. 427-432 ◽  
Author(s):  
Hatice Duzkale ◽  
Iman Jilani ◽  
Nada Orsolic ◽  
Ralph A. Zingaro ◽  
Mirna Golemovic ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Vitro Activity ◽  
Human Leukemia ◽  
Dimethylarsinic Acid ◽  
In Vitro Activity ◽  
Multiple Myeloma Cell

Inhibition of HIF1A Signaling by a Novel Class of Sulfonanilides for Targeted Treatment of Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2856-2856 ◽  
Author(s):  
Dirk Hose ◽  
Anja Seckinger ◽  
Hartmut Goldschmidt ◽  
Tobias Meiβner ◽  
Blanka Rebacz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Chromosomal Aberrations ◽  
Myeloma Cell ◽  
Proliferation Index ◽  
The Novel ◽  
Myeloma Cells ◽  
Signaling Inhibitors ◽  
Exposure Levels

Abstract Abstract 2856 Poster Board II-832 BACKGROUND. Molecular profiling of multiple myeloma allows the identification of novel targets, including HIF1A, and evaluation of their expression within large cohorts of patients. We report here the expression of HIF1A in myeloma and for the first time the preclinical testing of 4 members of a novel class of sulfonanilide HIF1A signaling inhibitors. PATIENTS AND METHODS. Expression of HIF1A was assessed using Affymetrix DNA-microarrays in 329 samples of CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive iFISH using a set of probes for the chromosomal regions 1q21, 6q21, 8p21, 9q34, 11q23, 11q13, 13q14.3, 14q32, 15q22, 17p13, 19q13, 22q11, as well as the translocations t(4;14)(p16.3;q32.3) and t(11;14)(q13;q32.3). Proliferation of primary myeloma cells (n=67) was determined by propidium iodine staining. The effect of the novel HIF1A signaling inhibitors ELR510490, ELR510454, ELR510444 and ELR105813 on the proliferation of 12 human myeloma cell lines and the first three on the survival of 5 primary myeloma cell-samples cultured within their microenvironment was tested, and their ability to inhibit HIF1A signaling was examined using a cell-based reporter assay. Studies were also conducted to determine in vitro stability (in plasma and microsomes), as well as single-dose PK (SDPK) parameters and maximum tolerated dose (MTD) levels after dosing in mice. RESULTS. We found (i) HIF1A to be expressed by 95.4% of CD138-purified primary myeloma cell samples from previously untreated patients. (ii) HIF1A expression shows a weak but significant correlation (r=0.3, p<0.001) with a gene expression based proliferation index. (iii) Of the chromosomal aberrations tested, myeloma cells of patients with presence of a translocation t(4,14) show a significantly higher expression of HIF1A (p<0.001) vs. patients without. Myeloma cells of hyperdiploid patients show a significantly lower expression of HIF1A (p=0.02) vs. non hyperdiploid patients. (iii) HIF1A expression does not show a correlation with event-free or overall survival. (iv) The sulfonanilides ELR510490, ELR510444, ELR510454 and ELR105813 completely inhibit proliferation of all tested myeloma cell lines at nM concentrations. (v) The compounds tested, i.e. ELR510490, ELR510444, ELR510454, are active on all primary myeloma cell-samples tested. (vi) The compounds show a pronounced effect on the HIF1A signaling pathway at EC50s of 1-25nM. (vii) Pre-clinical pharmacology data for the compounds ELR510444 and ELR510490 in mice indicate favorable absorption, distribution, metabolism, and excretion (ADME) profiles as well as exposure levels upon dosing at well-tolerated levels that are significantly above the in vitro EC50 in all the cell lines tested. CONCLUSION. HIF1A is expressed in almost all primary myeloma cells. The novel HIF1A signaling inhibitors tested are very active on myeloma cell lines as well as primary myeloma cells and show favorable in vivo profiles with exposure levels in mice significantly higher than the concentrations required for the inhibition of cell proliferation or apoptosis induction in vitro. This class of compounds thus represents a promising weapon in the therapeutic arsenal against multiple myeloma. Disclosures: Rebacz: ELARA Pharmaceuticals: Employment. Lewis:ELARA Pharmaceuticals: Employment. Schultes:ELARA Pharmaceuticals: Employment.

Constitutive and Immunoproteasome Inhibitors Increase IκB and Decrease NF-κB Expression In Dendritic Cell

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3493-3493
Author(s):  
Ahmad-Samer Samer Al-Homsi ◽  
Zhongbin Lai ◽  
Tara Sabrina Roy ◽  
Niholas Kouttab
Keyword(s):  
Multiple Myeloma ◽  
T Cell ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Peripheral Blood ◽  
Research Funding ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell

Abstract Introduction Constitutive and immunoproteasome inhibitors (C&IPI) were thought to suppress nuclear factor-κB (NF-κB) pathway by preventing IκB degradation, which prevents NF-κB translocation into the nucleus. This mechanism of action has since been questioned by a number of studies. First, bortezomib promoted constitutive NF-κB activity in endothelial cell carcinoma. Second, NF-κB constitutive activity was resistant to bortezomib in multiple myeloma cell lines. Third, bortezomib increased IκB mRNA but post-transcriptionally downregulated IκB in normal cells and in multiple myeloma cell lines resulting in induced canonical NF-κB activation. Lastly, bortezomib increased nuclear levels of IκB as opposed to lowering cytoplasmic levels in cutaneous T cell lymphoma cell line suggesting that nuclear translocation of IκB was possibly responsible for NF-κB inhibition. The inhibitory activity of C&IPI on dendritic cells (DC) is of interest in the prevention of graft versus host disease (GvHD). It has been shown that different C&IPI impede DC maturation and T cell priming both in vitro and in vivo. Herein we sought to understand the mechanism of action of proteasome and immunoproteasome inhibitors on DC and to test their effect on IκB and NF-IκB expression. Materials and Methods We first performed RT PCR on lysates of DC obtained from the peripheral blood of 7 patients who received post-transplant cyclophosphamide and bortezomib as prevention of GvHD on a phase I clinical trial. Patients received allogeneic transplantation from matched-related or unrelated donors. Patients received no other immunosuppressive therapy except for rabbit anti-thymocyte globulin for those receiving graft from unrelated donor. Steroids were not allowed on the study. Samples were obtained on days +1, +4, and +7. The results were analyzed in comparison to samples obtained on day 0 before stem cell infusion. We then performed the same experiment on lysates of DC obtained from the peripheral blood of healthy volunteer donors. DC were untreated or incubated with bortezomib (10 nM for 4 h), carfilzomib (30 nM for 1 h), oprozomib (100 nM and 300 nM for 4 h), ONX 0914 (200 nM for 1 h), PR-825 (125 nM for 1 h), or PR-924 (1000 nM for 1 h). The drug concentration and duration of exposure were chosen based on the IC50 on proteasome activity and to reproduce in vivo conditions. We also performed IκB western blot on DC isolated from peripheral blood of healthy volunteers, untreated or incubated with bortezomib (10 nM for 4 h) or oprozomib (300 nM for 4 h). Each experiment was performed at least in triplicate. Results We found that the combination of cyclophosphamide and bortezomib significantly and progressively increased IκB mRNA while decreasing NF-κB mRNA in DC studied ex vivo. We also found that all studied C&IPI increased IκB mRNA to a variable degree while only oprozomib (300 nM) decreased NF-κB mRNA in DC in vitro. Finally, both bortezomib and oprozomib increased IκB protein level in DC in vitro (figure). Conclusion Our data suggest that C&IPI increase IκB expression in DC. As opposed to the previously reported data in other cell types, the effect is not associated with post-transcriptional downregulation. Cyclophosphamide and bortezomib also decrease NF-κB expression in DC in vivo while only oprozomib had the same effect in vitro. The effect of C&IPI on IκB and NF-κB expression may represent a new mechanism of action and suggests their effect may be cell-type dependent. Disclosures: Al-Homsi: Millennium Pharmaceuticals: Research Funding. Off Label Use: The use of cyclophosphamide and bortezomib for GvHD prevention. Lai:Millennium Pharmaceuticals: Research Funding.

Mcl-1 Is Overexpressed in Multiple Myeloma and Correlates with Disease Severity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1419-1419
Author(s):  
Soraya Wuilleme-Toumi ◽  
Nelly Robillard ◽  
Patricia Gomez-Bougie ◽  
Philippe Moreau ◽  
Steven Le Gouill ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Disease Severity ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Lymphocytic Leukemia ◽  
Myeloma Cells

Abstract Multiple Myeloma (MM) is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. The expression of the pro-survival members of the Bcl-2 family has been shown to be a key process in the survival of myeloma cells. More particularly, Mcl-1 expression turned out to be critical for their survival. Indeed, knockdown of Mcl-1 by antisenses induces apoptosis in myeloma cells. Finally, Mcl-1 was found to be the only anti-apoptotic Bcl-2 family member which level of expression was modified by cytokine treatment of myeloma cells. For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC) i.e., myeloma cells from 55 patients with MM and 20 human myeloma cell lines using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. Forty-seven percent of patients with MM at diagnosis (p=.017) and 80% at relapse (p=.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only myeloma cell lines but not reactive plasmocytoses have abnormal Mcl-1 expression, although both plasmocyte expansion entities share similar high proliferation rates (&gt;20%). Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. This shows that the overexpression of Mcl-1 is clearly related to malignancy rather than to proliferation. It will be important to know whether the overexpression of Mcl-1 is related to an abnormal response to cytokines like Interleukin-6 or to mutations of the promoter of the Mcl-1 gene as already described in B chronic lymphocytic leukemia. Finally, level of Mcl-1 expression is related to disease severity, the highest values being correlated with the shortest event-free survival (p=.01). In conclusion, Mcl-1 which has been shown to be essential for the survival of human myeloma cells in vitro is overexpressed in vivo in MM and correlates with disease severity. Mcl-1 represents a major therapeutical target in MM.

Upregulation of MMP-2 & 9 by Co-Culture of Human Myeloma Cell Lines and Endothelial Cells May Be Responsible for Protection Against Cytotoxic Drugs.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5080-5080
Author(s):  
Shankaranarayana Paneesha ◽  
Raghu Adya ◽  
Hemali Khanji ◽  
Ed Leung ◽  
C. Vijayasekar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Mrna Expression ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Human Myeloma Cell

Abstract Multiple myeloma is a clonal lymphoproliferative disorder characterised by the proliferation of plasma cells in the bone marrow. Inspite of good initial response, it is associated with universal relapse. We hypothesise this is due to sanctuary provided to myeloma cells by the endothelium. Matrix metalloproteinases (MMPs) are shown play a role in cell growth, invasion, angiogenesis, metastasis and bone degradation. We show here the protection offered by endothelial cells to human myeloma cell lines in in-vitro co-culture with upregulation of MMP-2 & 9 and the role of GM6001 MMP inhibitor (Ilomastat) in overcoming this protection. Human myeloma cell lines (H929, RPMI 8226, U266 & JJN3) with or without endothelial cells (human umbilical vein endothelial cells and EaHy 926 cell line) in-vitro co-culture were treated with melphalan, dexamethasone, arsenic trioxide and Ilomastat. Cytotoxicity/proliferation were assessed by the alamarBlue™ assay (Serotec) and validated by Annexin V-FITC apoptosis detection Kit (Calbiochem) and BrDU proliferation assay (BD Pharmingen™). Gelatin Zymography was used to demonstrate activity of MMP-2 & 9 in the supernatant. MMP-2 and 9 mRNA expression was quantified by Real Time Quantitative PCR (ROCHE). Co-culture of human myeloma cell lines with endothelial cells lead to increase in the proliferation of myeloma cell lines and also protected them from the cytotoxicity of chemotherapeutic agents. MMP-2 & 9 activity was upregulated by the co-culture. MMP-2 mRNA expression in human myeloma cell lines increased following 4 hr co-culture. Treatments with Ilomastat lead to the suppression of proliferation in co-culture in a dose dependent manner, associated with a reduction of MMP-2 and 9 activity. Our study shows endothelial cells offer protection to human myeloma cell lines in the presence of cytotoxic agents. This may result in the sanctuary of myeloma cells in bone marrow leading to ultimate relapse of disease. Our study also demonstrates the upregulation of MMP-2 and 9 by co-culture and increased cytotoxicity achieved by the inhibition of MMPs. Further studies are needed to determine the exact role of MMPs in myeloma biology as MMP inhibition may be an interesting therapeutic target and help in averting relapse in multiple myeloma.

A Novel Mouse Model of Multiple Myeloma Representative of Human Disease and Its Use in Preclinical Therapeutic Assessment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2907-2907
Author(s):  
Rosemary A Fryer ◽  
Timothy J Graham ◽  
Emma M Smith ◽  
Brian A Walker ◽  
Gareth J Morgan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Human Condition ◽  
Myeloma Cells ◽  
Treatment Groups ◽  
Hyperintense Signal

Abstract Abstract 2907 In order to aid the pre-clinical development of novel therapeutics for multiple myeloma, an in vivo model which recapitulates the human condition in particular tumor growth patterns and response to treatment is required. An important feature of such a model is the interaction of the myeloma cells with the bone marrow microenvironment as this is known to modulate tumor activity and protect against drug-induced apoptosis. We have developed a model with myeloma restricted to the bone marrow, which proceeds rapidly from initial inoculation to disease progression, and possesses a range of chemo-sensitive markers with which to monitor anti-tumor response. Female NOD/SCID γcnull mice were injected inta-osseously with luciferase-tagged myeloma cell lines. Disease progression was monitored weekly by bioluminescent imaging (BLI) and measurement of paraprotein levels (ELISA). These methods were compared to histological assessment of tumor infiltration and MRI which provided a quantitative measurement of progression. On T2-weighted images tumor was identified as a hyperintense signal enclosed within cortical bone. Tumor burden was quantified from regions of interest drawn on the periphery of the hyperintense signal. Luciferase-tagged cells engrafted by 3 weeks at the injection site and progressed to the femurs, spine and pelvis from week 4. BLI showed a significant increase in radiance from 5.6×105 to 43.0×105p/s/cm2/sr between weeks 5 and 7 (p<0.05). Quantification of tumor volume by MRI showed a significant increase from 6.4mm3 to 27.6mm3 between weeks 4 and 8 (p<0.05) and μCT demonstrated lytic disease. Serum levels of Igλ increased from 860ng/ml to 4325ng/ml during this period (p<0.05), which mirrored the changes seen with BLI and MRI. Flow cytometry and histology confirmed the confinement of CD138 positive myeloma cells within the bone. These results indicate successful engraftment of human myeloma cell lines with induction of myeloma in a pattern similar to the human condition. We have adapted this model to study primary patient material. 10 mice were implanted with samples from 3 cases of plasma cell leukemia with complex cytogenetics. 5 of these developed myeloma confined to the bone marrow, 2 with additional plasmacytoma localized at the injection site, over a period of 1–5months. We have characterized the original patient cells with gene expression, SNP based gene mapping and have characterized the nature of the engrafted cells using similar technology. We have also shown the model is suitable for preclinical assessment of anti-myeloma agents using bortezomib and a novel aminopeptidase inhibitor, tosedostat (CHR-2797). Non-treated mice displayed a significant increase in radiance from 16.13×105 to 69.00×105p/s/cm2/sr (p<0.01). In comparison, in the bortezomib and tosedostat treated groups no significant increase in radiance was seen (bortezomib: 5.22×105 to 1.12×105 p/s/cm2/sr; tosedostat: 9.92×105 to 13.78×105p/s/cm2/sr). Paraprotein levels mimicked these changes in BLI. At the end of treatment Igλ levels in control, bortezomib and tosedostat treated mice were 2473.7, 132.5 and 923.0ng/ml, respectively. Igλ levels in both treatment groups were significantly different from control (p<0.001). Average tumor volumes derived from MRI were significantly different in bortezomib (14.7mm3) and tosedostat treated (23.4mm3) groups compared to non-treatment (33.0mm3). The volumes for the bortezomib treated group showed no significant difference from control mice. In addition, there was a decrease in CD138 expression by flow cytometry in bone aspirates from treatment groups compared to control which was mirrored in histological samples. In conclusion using both myeloma cell lines and primary patient cells, we have developed a model which recapitulates human myeloma with secretion of paraprotein, disease confined to the bone marrow, lytic bone lesions and spinal compression. In addition, this model is suitable for assessing the efficacy of novel therapeutics in vivo, using a number of non-invasive tumor markers such as BLI and MRI. Disclosures: Morgan: J&J: Honoraria, Speakers Bureau. Davies:J&J: Honoraria, Speakers Bureau.

Sphingolipids As a Novel Target For The Treatment Of Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3163-3163 ◽  
Author(s):  
Jagadish Kummetha Venkata ◽  
Robert K Stuart ◽  
Luciano J Costa ◽  
Ningfei An ◽  
Houjian Cai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Myeloma Cell ◽  
Sphingolipid Metabolism ◽  
Myeloma Cells ◽  
S1p Receptors

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.

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