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

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.

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Inhibition of Aurora-Kinases for Tailored Risk-Adapted Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v112.11.248.248 ◽

2008 ◽

Vol 112 (11) ◽

pp. 248-248

Author(s):

Dirk Hose ◽

Thierry Rème ◽

Tobias Meißner ◽

Jérôme Moreaux ◽

Anja Seckinger ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Chromosomal Aberrations ◽

Genetic Instability ◽

Kinase Inhibitor ◽

Aurora Kinase ◽

Myeloma Cell ◽

High Dose ◽

Aurora A ◽

Myeloma Cells

Abstract BACKGROUND. At the time of diagnosis, myeloma cells are characterized by a low proliferation rate that increases in relapse. Presence of proliferation correlates with adverse prognosis. At the same time, myeloma cells harbor a high median number of chromosomal aberrations, often associated with genetic instability. Cellular proliferation and genetic instability in turn have been associated with Aurora-kinase expression in several cancer entities, including multiple myeloma. PATIENTS AND METHODS. Expression of Aurora-A, -B and -C was assessed using Affymetrix DNA-microarrays in 784 samples including two independent sets of 233 and 345 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 clinical Aurora-kinase inhibitor VX680 on proliferation of 20 human myeloma cell lines and survival of 5 primary myeloma cell-samples was tested. RESULTS. We found Aurora-A and -B to be expressed at varying frequencies in primary myeloma cells of different patient-cohorts, including 23% for Aurora A in our first cohort of patients treated with high dose therapy (see figure shown below). Aurora-C expression was found in testis-samples only. Myeloma cell-samples with detectable Aurora-A expression show a significantly higher proliferation rate, whereas the number of chromosomal aberrations (aneuploidy) is not higher compared to myeloma-cells with absent Aurora-A expression. The same holds true for subclonal aberrations (i.e. genetic instability). The Aurora-kinase inhibitor VX680 induces apoptosis in all myeloma cell lines and primary myeloma cell-samples tested. Presence of Aurora-A expression delineates significantly inferior event-free and overall survival in two independent cohorts of patients undergoing high-dose chemotherapy and autologous stem cell transplantation. This observation is independent of conventional prognostic factors, i.e. serum-ß2-microglobulin or ISS-stage. CONCLUSION. Aurora-kinase inhibitors (including VX680 tested here) are very active on myeloma cell lines as well as primary myeloma cells and represent a promising weapon in the therapeutic arsenal against multiple myeloma. Gene expression profiling allows the assessment of Aurora-kinase expression and thus in turn a tailoring of treatment to patients expressing Aurora-A associated with adverse prognosis. Figure Figure

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

Blood ◽

10.1182/blood.v104.11.1419.1419 ◽

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 (>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.

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

Blood ◽

10.1182/blood.v106.11.1581.1581 ◽

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.

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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 ◽

10.1182/blood.v108.11.5080.5080 ◽

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.

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

Blood ◽

10.1182/blood.v114.22.2848.2848 ◽

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.

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Sphingolipids As a Novel Target For The Treatment Of Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.3163.3163 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3163-3163 ◽

Cited By ~ 1

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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Melphalan and XPO1 Inhibitor Combination Therapy for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.2084.2084 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2084-2084 ◽

Cited By ~ 1

Author(s):

Joel G Turner ◽

Jana L Dawson ◽

Steven Grant ◽

Kenneth H. Shain ◽

Yun Dai ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

De Novo ◽

Ex Vivo ◽

Myeloma Cell ◽

Drug Resistant ◽

Cell Viability Assay ◽

Myeloma Cells ◽

Multiple Myeloma Cell

Abstract Introduction High-dose melphalan chemotherapy with autologous stem cell transplant remains the standard of care for the treatment of multiple myeloma. However, patients eventually develop drug resistance and die from progressive disease despite the introduction of therapies using proteosome inhibitors (PIs) and immunomodulatory drugs (IMIDs). The incurable nature of multiple myeloma clearly demonstrates the need for novel agents and treatments. Here, our aim was to investigate whether the use of XPO1 (exportin 1, CRM1) inhibitors (XPO1i) could sensitize de novo and acquired drug-resistant multiple myeloma cells both in vitro and ex vivo to the alkylating agent melphalan. Materials and Methods Human multiple myeloma cell lines NCI-H929, RPMI-8226, U266 and PBMC controls were treated in vitro with the XPO1i KOS-2464 and the orally available Selective Inhibitor of Nuclear Export (SINE) selinexor (KPT-330) or) +/- melphalan. Multiple myeloma cells were grown at high-density conditions (>3-5x106 cells/mL). High-density multiple myeloma cells have been shown to possess de novo drug resistance. Sensitivity of the XPO1i/melphalan-treated NCI-H929 cells was measured by cell viability assay (CellTiter-Blue). Apoptosis in XPO1i/melphalan-treated NCI-H929, RPMI-8226, and U266 cells was assayed using flow cytometry (activated caspase 3). Proximity ligation assays were performed to assess XPO1-p53 binding in the presence of an XPO1i. Western blots of XPO1i-treated myeloma cells were performed for nuclear and total p53. Drug-resistant U266 (PSR) and 8226 (8226/B25) myeloma cell lines were developed by incremental exposure to bortezomib. PSR cells are able to grow in 15 nM bortezomib and the 8226/B25 in 25 nM. These resistant myeloma cells were treated in vitro with XPO1i +/- melphalan. Sensitivity to therapy was measured by apoptosis and cell viability assay. Multiple myeloma cells isolated from patients with newly diagnosed, relapsed, or refractory disease were treated with XPO1i +/- melphalan and CD138+/light chain+ myeloma cells and assayed for apoptosis. Results Multiple myeloma cell (NCI-H929) viability was decreased synergistically by XPO1i when used in combination with melphalan, as shown by the calculated combinatorial index (CI) values. We examined sequencing of the drugs and found that concurrent treatment with melphalan (10 µM) and selinexor (300 nM) for 48 hours produced the best results (CI value 0.370, n=6). Sequential treatment (selinexor for 24 hours followed by melphalan for an additional 24 hours) or the reverse sequence had slightly less synergy, with CI values of 0.491 (n=9) and 0.565 (n=3), respectively. Normal PBMC control cells were unaffected by XPO1i/melphalan treatment as shown by viability and apoptotic assays. Proximity ligation assay demonstrated that XPO1i blocks XPO1/p53 binding. Western blot showed that the XPO1i treatment of myeloma cells increased nuclear and total p53. Drug-resistant 8226/B25 myeloma cells but not PSR cells were found to be resistant to melphalan when compared to parental cell lines. Both resistant myeloma cell lines were sensitized by XPO1i to melphalan as shown by apoptosis assay (3- to 10-fold). CD138+/light chain+ myeloma cells derived from newly diagnosed, relapsed, and refractory myeloma patients were also sensitized by XPO1 inhibitors to melphalan as demonstrated by apoptotic assays (e.g. activated caspase 3). Conclusions XPO1i synergistically improved the response of de novo and acquired drug-resistant myeloma cells to melphalan in vitro and ex vivo. It is possible that this synergy may be due to an increase of nuclear p53 by XPO1i and the reported activation of p53 by melphalan. Future studies include in vitro experiments using drug-resistant human U266 myeloma cells in NOD-SCID-gamma mice and clinical trials using melphalan in combination with the SINE selinexor. Combination therapies using selinexor and melphalan may significantly improve the treatment of myeloma. Disclosures Kauffman: Karyopharm Therapeutics: Employment. Shacham:Karyopharm Therapeutics: Employment.

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In Vitro Activity of a Novel Organic Arsenical (S-Dimethylarsino-Glutathione, ZIO-101) Against Multiple Myeloma.

Blood ◽

10.1182/blood.v106.11.5163.5163 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5163-5163 ◽

Cited By ~ 1

Author(s):

Lawrence H. Boise ◽

Alejo A. Morales ◽

Delia Gutman ◽

Kelvin P. Lee

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Phase I ◽

Cell Lines ◽

Myeloma Cell ◽

Differential Sensitivity ◽

Response Curves ◽

Myeloma Cells ◽

Highly Active

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.

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1′-Acetoxychavicol Acetate (ACA) Is a Novel NF-κB Inhibitor with Significant Activity Against Multiple Myeloma in Vitro and in Vivo.

Blood ◽

10.1182/blood.v104.11.765.765 ◽

2004 ◽

Vol 104 (11) ◽

pp. 765-765 ◽

Cited By ~ 2

Author(s):

Keisuke Ito ◽

Tomonori Nakazato ◽

Yoshitaka Miyakawa ◽

Ming Ji Xian ◽

Taketo Yamada ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Myeloma Cell ◽

Serine Phosphorylation ◽

Significant Activity ◽

Dependent Manner ◽

Myeloma Cells ◽

Induced Apoptosis

Abstract 1′-acetoxychavicol acetate (ACA) is a component of traditional Asian condiment, obtained from rhizomes of the commonly used ethno-medicinal plant Languas galanga (Zingiberacetate). Recent extensive studies revealed that ACA has potent chemopreventive effects against various tumors. More recently, we have reported that ACA induces apoptosis of myeloid leukemic cells via mitochondrial- and Fas-mediated dual pathway. The transcription factor NF-κB confers significant survival potential in myeloma cells; therefore, it has emerged as a therapeutic target for the treatment of multiple myeloma. Multiple myeloma is an incurable hematological disorders, which has been fatal outcome despite of high dose chemotherapy with stem cell transplantation; therefore, a novel biologically based therapeutic approach is desired. In this study, we investigated the effects of ACA on myeloma cells in vitro and in vivo, and further examined the molecular mechanisms of ACA-induced apoptosis in myeloma cells. ACA dramatically inhibited cellular growth of various human myeloma cell lines (RPMI8226, U266, IM9, and HS-Sultan) as well as freshly isolated myeloma cells from patients, but not normal bone marrow cells, in a dose (0-20 μM)- and time (0-24 h)-dependent manner. Cultivation with 10 μM ACA rapidly increased the population of cells in the G0/G1 phase with a reduction of cells in the S phase, and a strong induction of apoptosis was shown by the appearance of a hypodiploid DNA peak with sub-G1 DNA content 3 h after treatment. Treatment with ACA induced both caspase-3, -9, and caspase-8 activities, suggesting that ACA-induced apoptosis in myeloma cells mediates both mitochondrial- and Fas-dependent pathways. Furthermore, we investigated the effects of ACA on NF-κB activity in myeloma cells, and were able to demonstrate that ACA significantly inhibited serine phosphorylation and degradation of IκBα in a time-dependent manner. ACA rapidly decreased the nuclear expression of NF-κB, but increased the accumulation of cytosol NF-κB in RPMI8226 cells, indicating that ACA inhibits translocation of NF-κB from the cytosol to the nucleus. In addition, we also confirmed the inhibitory effects of ACA on NF-κB activation by ELISA in myeloma cell lines and fresh samples. ACA had a synergistic proapoptotic effect with another NF-κB inhibitor, MG-132 and TLCK. In contrast, NF-κB activator, PMA, dramatically abrogated ACA-induced apoptosis in myeloma cells. These in vitro studies prompted us to examine whether the effects of ACA are equally valid in vivo. To evaluate the effects of ACA in vivo, RPMI8226-transplanted NOD/SCID mice were treated with ACA. Tumor weight decreased in the mice that were injected ACA (mean weight: 0.04±0.06 g in the ACA-treated group vs. 0.63±0.29 g in the control group; p<0.01). During the treatment, ACA-treated mice appeared healthy, and pathological analysis at autopsy revealed no ACA-induced tissue changes in any of the organ, indicating that ACA might be developed as a new potent anti-cancer agent for the management of multiple myeloma. In conclusion, ACA has an inhibitory activity of NF-κB, and induces apoptosis of myeloma cells in vitro and in vivo. Therefore, ACA provides the new biologically based therapy for the treatment of multiple myeloma patients as a novel NF-κB inhibitor.

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

Blood ◽

10.1182/blood.v106.11.5190.5190 ◽

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.

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