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.

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.

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

Thymoglobulin (Polyclonal Rabbit Anti Thymocyte Globulin) Has In Vivo Activity in a Mouse Model of Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3487-3487
Author(s):  
Michael Timm ◽  
Linda Wellik ◽  
Teresa Kimlinger ◽  
Jessica Haug ◽  
Michael Kline ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Growth ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Solid Organ ◽  
Polyclonal Rabbit

Abstract Background: Multiple myeloma remains incurable with current approaches and newer therapies are needed to improve the outcome of these patients. While monoclonal antibody based therapies have been successful in some of the hematological malignancies, such approaches have had limited efficacy in the setting of myeloma. Thymoglobulin (polyclonal rabbit antithymocyte globulin, Genzyme) (Thymo) has been extensively evaluated in the setting of allogeneic blood and marrow transplantation and solid organ transplants. Given the polyclonal nature of this product, with antibodies against different B cell antigens, we evaluated the in vitro and in vivo activity of Thymo in 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. Apoptosis was measured by flow cytometry using Annexin V/PI in cell lines and Apo 2.7 in primary patient plasma cells. Shifts in expression of a variety of different B cell and plasma cell antigens were examined on several different myeloma cell lines following Thymo treatment in order to identify the potential antigenic targets. In vivo activity of thymo was evaluated in a SCID plasmacytoma model injected with RPMI myeloma cell lines. 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 with LC50 of around 1 mg/mL. Additionally, thymo was cytotoxic MM cell lines resistant to conventional agents such as doxorubicin (Dox40), melphalan (LR5) and dexamethasone (MM1R). Thymo induced apoptosis in MM cell lines and in patient derived primary myeloma cells. When tested in combination with other anti-myeloma agents an additive effect was seen with doxorubicin, PS341 and melphalan. Using competitive flow cytometry, we identified CD138, CD38, Cd45, CD126, CD49d (VLA4), as well as CD20 as antigens likely to be targeted by Thymo. Tumor bearing mice injected with Thymo at two different doses (5 mg/kg and 10 mg/kg for five days) had significantly delayed tumor growth compared to non-injected mice, and this translated into a better survival for these mice. Mice receiving 10 mg/kg dose had a slower tumor growth compared to 5 mg/kg dose (Figure). Conclusions: Thymoglobulin has promising in vitro and in vivo activity in the setting of myeloma. These studies will provide the rational for future clinical development of this agent in myeloma alone or in combination with other agents. Based on these results, we are in the process of initiating a clinical trial combining Thymo with Melphalan. Figure Figure

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.

scholarly journals Pharmacologic Inhibition of the Histone Methyltransferase SETD2 with EZM0414 As a Novel Therapeutic Strategy in Relapsed or Refractory Multiple Myeloma and Diffuse Large B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1142-1142
Author(s):  
Jennifer Totman ◽  
Dorothy Brach ◽  
Vinny Motwani ◽  
Selene Howe ◽  
Emily Deutschman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Publicly Traded ◽  
The Past ◽  
Molecule Inhibitor

Abstract Introduction: SETD2 is the only known histone methyltransferase (HMT) capable of catalyzing H3K36 trimethylation (H3K36me3) in vivo. It plays an important role in several biological processes including B cell development and maturation, leading to the hypothesis that SETD2 inhibition in these settings could provide anti-tumor effects. The normal process of B cell development/maturation renders B cells susceptible to genetic vulnerabilities that can result in a dysregulated epigenome and tumorigenesis, including in multiple myeloma (MM) and diffuse large B-cell lymphoma (DLBCL). For example, 15%-20% of MM harbors the high risk (4;14) chromosomal translocation, resulting in high expression of the multiple myeloma SET domain (MMSET) gene. MMSET is an HMT that catalyzes H3K36me1 and H3K36me2 formation and extensive scientific work has established overexpressed MMSET as a key factor in t(4;14) myeloma pathogenesis. To the best of our knowledge MMSET has eluded drug discovery efforts, however, since t(4;14) results in high levels of the H3K36me2 substrate for SETD2, inhibiting SETD2 offers promise for targeting the underlying oncogenic mechanism driven by MMSET overexpression in t(4;14) MM patients. In addition, SETD2 loss of function mutations described to date in leukemia and DLBCL are always heterozygous, suggesting a haploinsufficient tumor suppressor role for SETD2. This observation points to a key role for SETD2 in leukemia and lymphoma biology and suggests that therapeutic potential of SETD2 inhibition may also exist in these or similar settings. EZM0414 is a first-in-class, potent, selective, orally bioavailable small molecule inhibitor of the enzymatic activity of SETD2. We explored the anti-tumor effects of SETD2 inhibition with EZM0414 in MM and DLBCL preclinical studies to validate its potential as a therapy in these tumor types. Methods: Cellular proliferation assays determined IC 50 values of EZM0414 in MM and DLBCL cell line panels. Cell line-derived xenograft preclinical models of MM and DLBCL were evaluated for tumor growth inhibition (TGI) in response to EZM0414. H3K36me3 levels were determined by western blot analysis to evaluate target engagement. Combinatorial potential of SETD2 inhibition with MM and DLBCL standard of care (SOC) agents was evaluated in 7-day cotreatment in vitro cellular assays. Results: Inhibition of SETD2 by EZM0414 results in potent anti-proliferative effects in a panel of MM and DLBCL cell lines. EZM0414 inhibited proliferation in both t(4;14) and non-t(4;14) MM cell lines, with higher anti-proliferative activity generally observed in the t(4;14) subset of MM cell lines. The median IC 50value for EZM0414 in t(4;14) cell lines was 0.24 μM as compared to 1.2 μM for non-t(4;14) MM cell lines. Additionally, inhibitory growth effects on DLBCL cell lines demonstrated a wide range of sensitivity with IC 50 values from 0.023 μM to &gt;10 μM. EZM0414 resulted in statistically significant potent antitumor activity compared to the vehicle control in three MM and four DLBCL cell line-derived xenograft models. In the t(4;14) MM cell line-derived xenograft model, KMS-11, robust tumor growth regressions were observed at the top two doses with maximal TGI of 95%. In addition, two non-t(4;14) MM (RPMI-8226, MM.1S) and two DLBCL xenograft models (TMD8, KARPAS422) demonstrated &gt; 75% TGI; with two additional DLBCL models (WSU-DLCL2, SU-DHL-10) exhibiting &gt; 50% TGI in response to EZM0414. In all models tested, the antitumor effects observed correlated with reductions in intratumoral H3K36me3 levels demonstrating on-target inhibition of SETD2 methyltransferase activity in vivo. In vitro synergistic antiproliferative activity was also observed when EZM0414 was combined with certain SOC agents for MM and DLBCL. Conclusions: Targeting SETD2 with a small molecule inhibitor results in significantly reduced growth of t(4;14) MM, as well as non-t(4;14) MM and DLBCL cell lines, in both in vitro and in vivo preclinical studies. In addition, in vitro synergy was observed with EZM0414 and certain SOC agents commonly used in MM and DLBCL, supporting the combination of SETD2 inhibition with current MM and DLBCL therapies. This work provides the rationale for targeting SETD2 in B cell malignancies such as MM, especially t(4;14) MM, as well as DLBCL, and forms the basis for conducting Phase 1/1b clinical studies to evaluate the safety and activity of EZM0414 in patients with R/R MM and DLBCL. Disclosures Totman: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Brach: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Motwani: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Howe: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Deutschman: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Lampe: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Riera: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Tang: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Eckley: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Alford: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Duncan: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Farrow: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Dransfield: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Raimondi: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Thomeius: Foghorn Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cosmopoulos: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Kutok: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company.

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

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

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

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.

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.

Canonical NF-kB Pathway Is Not Constitutively Activated and May Not Be the Major Bortezomib Target in Mantle Cell Lymphoma (MCL).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2591-2591
Author(s):  
Valerie Camara-Clayette ◽  
Yves Lepelletier ◽  
Ivan Moura ◽  
William Vainchenker ◽  
Olivier Hermine ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Dominant Negative ◽  
In Vitro Assays ◽  
Lymphoid Malignancies ◽  
Empty Vector ◽  
Gene Assay

Abstract Bortezomib, a proteasome inhibitor, has promising activity in MCL. Bortezomib mechanism of action is complex since multiple proteins and cellular pathways are regulated by proteasome degradation. By blocking the degradation of IKBa, the canonical NF-kB pathway is one of the major Bortezomib target in lymphoid malignancies such as multiple myeloma. NF-kB activation has not been extensively assessed in other lymphoid malignancies such as MCL. NF-kB complexes (heterodimers P50/P65) were assessed by EMSA assays in REC GRANTA 519, NCEB, JEKO, JUN and UPN1 MCL cell-lines. EBV was present in 3/6 cell lines. P50/P65 complexes were detected in all EBV positive cell lines and only in 1/3 EBV negative cell-lines (JEKO). No nuclear P65 protein was detected by immunofluorescence or Western-blot analysis in the 2 other negative EBV cell lines (REC and UPN1). NF-kB transcriptional activity was measured by a luciferase-based reporter gene assay. Spontaneous NF-kB activity was low in UPN1, REC and JEKO compared to GRANTA 519 EBV positive cell-line (3 to 4 time lower) but can be strongly activated (up to 10 fold) using MEKK cotransfection assay. This suggest that NF-kB is functional, but not constitutively activated. Stable infections with Migr1-IRES-GFP IkBM (dominant negative IKB) or empty vector, were performed in GRANTA 519 EBV positive as well as in UPN1 cell line. No differences in proliferation or apoptosis were observed in UPN1 stably infected with IkBM or with the empty vector but GRANTA 519 EBV positive showed increased apoptosis and proliferation inhibition when infected with IkBM. Likewise, no P50/P65 heterodimers complexes were detected by EMSA in 4 patients with MCL. In vitro assays showed that UPN1 and JEKO cell lines had comparable sensitivities to Bortezomib than Multiple myeloma cell lines reported in the literature (IC 50: 6 nM and 12 nM respectively). This was also true for the 3 patients lymphoma cells assessed in vitro (12 nM). Taken together these results suggest that canonical NF-kB activation pathway is not constitutively active in EBV negative MCL cell lines and patients samples and could be associated with EBV infection in some MCL cell-lines. Therefore, this strongly suggests that Bortezomib target other molecules in MCL.

Cyclin-Dependent Kinase Inhibitor Seliciclib Shows In Vitro Activity in Diffuse Large B Cell Lymphomas.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4738-4738
Author(s):  
Francesco Bertoni ◽  
Katia Lacrima ◽  
Andrea Rinaldi ◽  
Sara Vignati ◽  
Vittoria Martin ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Active Agent ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Large B Cell

Abstract Background. Despite recent improvements in treatment, a significant fraction of patients with diffuse large B cell lymphoma (DLBCL) still fail therapy. Therefore, new therapeutic modalities are needed to advance the cure rate. Seliciclib (CYC202, R-roscovitine) is a purine analogue developed as an inhibitor of CDK2/cyclin E CDK7/cyclin H and CDK9/cyclin T. Seliciclib has been shown to be active in B cell neoplasms, such as mantle cell lymphoma, chronic lymphocytic leukemia and in multiple myeloma in vitro. The aim of this study was to assess the in vitro activity of seliciclib in DLBCL. Materials and methods. The anti-proliferative activity of seliciclib was tested in nine human DLBCL cell lines and six DLBCL primary cell cultures. The effects of seliciclib on the cell cycle and on apoptosis, as well as on transcription-related proteins were assessed. Results. The cell viability of all DLBCL cell lines and primary cells was reduced by seliciclib treatment. The IC50 for the cell lines ranged from 13 to 36 μM. The effect of seliciclib was independent of the genetic aberrations characterizing the cell lines. After seliciclib exposure cells accumulated in G2/M or in G1 phase, with most of the cells showing signs of apoptosis. Despite the clear cytotoxic effect and induction of apoptosis, we could not identify a unique mechanism of action. Conclusions. Our in vitro data suggest that seliciclib is an active agent in DLBCL. Its efficacy is apparently independent of the underlying chromosomal translocations characteristic of DLBCL. The drug might represent a new therapeutic agent in this lymphoma subtype.

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