The Sequence-Selective DNA Cross-Linking Agent SJG-136 (SG2000, BN2629) Is Highly Potent in Multiple Myeloma Cells and Is Synergistic with Bortezimib

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5166-5166
Author(s):  
Elisabeth J Walsby ◽  
Alan Burnett ◽  
Steven Knapper ◽  
Chris Fegan ◽  
Chris Pepper
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Single Agent ◽  
Annexin V ◽  
Cross Linking ◽  
Incurable Disease ◽  
High Level

Abstract Despite recent advances in the treatment of multiple myeloma (MM), including the introduction of bortezimib, it remains an incurable disease with a short median survival. The development of new agents with efficacy in MM therefore remains both important and urgent. SJG-136 (SG2000, BN2629) is a novel DNA cross-linking agent that binds in a sequence-selective manner in the minor groove of the DNA helix. It is structurally novel compared with other clinically used DNA cross-linking agents and has exhibited a unique pattern of activity in the NCI 60-cell line screen. We have previously shown that this agent is highly effective against primary chronic lymphocytic leukaemia cells. In this study we evaluated its potential as a therapy for MM in cell lines and plasma cells derived from patients. The MM cell lines H929, U266 and JJN3 were shown to have a mean LD50 of 5.1nM (±3.4) following in vitro culture with SJG-136 for 48 hours. This was accompanied by a dose-dependent increase in the percentage of apoptotic cells as evidenced by Annexin V positivity and caspase-3 activation. In comparison to the standard therapies for MM, SJG-136 demonstrated significantly lower LD50 values than doxorubicin (P=0.004), Melphalan (P=0.004) and bortezimib (P=0.03). We also assessed the ability of SJG-136 to kill plasma cells from primary myeloma samples (identified by CD38 and CD138 positivity). These cells were sensitive to SJG-136 with a mean LD50 of 2.3nM (± 0.96) following 48 hour exposures to SJG-136. In contrast, normal bone marrow was significantly less affected by SJG-136 than the myeloma samples (P<0.0001). Finally, we assessed the potential for synergy between SJG-136 and these standard treatments using the H929 cell line as a model. SJG-136 was combined with doxorubicin (1:125), melphalan (1:4000) and bortezimib (1:8) and the combination index (CI) was calculated to assess synergy. A CI of less than 1 was considered synergistic. We found no evidence for synergy between SJG-136 and doxorubicin or melphalan (CI=1.29 and 1.75 respectively). However, SJG-136 was synergistic with bortezimib (CI=0.47) possibly reflecting their different mechanisms of action. In conclusion, SJG-136 is a potentially valuable addition to the battery of drugs available for treatment of MM. Not only was SJG-136 highly potent as a single agent in MM cell lines and primary plasma cells but it also showed a high level of synergy with the proteasome inhibitor bortezimib.

ABT-737, an Inhibitor of Bcl-2/Bcl-XL Proteins, Is Cytotoxic in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1589-1589
Author(s):  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
John A. Lust ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Cell Lines ◽  
Plasma Cells ◽  
Annexin V ◽  
Significant Activity ◽  
Rpmi 8226 ◽  
Dose Dependent

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation. 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 of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA) Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail. Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

Role of c-Kit in Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5047-5047
Author(s):  
Juan Carlos Montero ◽  
J.F. San Miguel ◽  
Atanasio Pandiella
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Mammalian Target Of Rapamycin ◽  
Retroviral Infection ◽  
Extracellular Region ◽  
Survival Pathways ◽  
High Level ◽  
Therapeutic Compounds

Abstract C-Kit is a receptor tyrosine kinase whose activation occurs by interaction with its ligand, the Stem Cell Factor (SCF). Two distinct isoforms of c-Kit have been identified, and are characterized by the presence or absence of the tetrapeptide sequence Gly-Asn-Asn-Lys (GNNK) in the juxtamembrane domain of the extracellular region. These isoforms signal with distinct properties. c-Kit has been reported to be expressed in the plasma cells of about 20% of patients with Multiple myeloma (MM), but not in normal plasma cells. Yet, its functional and biochemical signalling properties in myeloma cells have not been described. In this study we show that patients and cell lines of MM co-expressed both c-Kit isoforms (GNNK+ and GNNK−), being predominant the GNNK− isoform. c-Kit was activated by SCF in the cell line RPMI8226, and in cell lines derived from patients. To study the properties of the two c-Kit isoforms, we have expressed these isoforms in the cell line MM1S which do not express these receptors in a significantly high level. Both receptors types were expressed by retroviral infection. Addition of SCF provoked their tyrosine phosphorylation. Imatinib mesylate, that is a known inhibitor of c-Kit, inhibited the phosphorylation induced by SCF of both isoforms. Both receptors coupled to the PI3K/Akt pathway, but were unable to substantially activate the Erk1/2 MAPKs. Differences in the kinetics of phosphorylation between the isoforms GNNK+ and GNNK− were observed. The variant GNNK− was more rapidly and highly phosphorylated than the GNNK+ isoform, which was activated more slowly but this activation persisted a longer time. In MM1S-GNNK+ cells the principal substrate of Akt activated by SCF was the p70S6K, and this activation was dependent on the activity of the mammalian Target Of Rapamycin (mTOR). Finally, the expression of the c-Kit isoforms in MM1S partially prevented their death induced by dexamethasone. These data indicate that c-Kit expression in MM cells is functional and couples to survival pathways that may modulate cell death in response to therapeutic compounds used in the treatment of this disease.

Combination of Akt/PKB Inhibition (Perifosine) and Farnesyl Transferase Inhibition (Tipifarnib) Results in Increased Cell Death in Myeloma Cells Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1568-1568 ◽  
Author(s):  
Rajni Sinha ◽  
Ebenezer David ◽  
Emily Zeilter ◽  
Claire Torre ◽  
Jonathan L. Kaufman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Death ◽  
Combination Therapy ◽  
Cell Line ◽  
Cell Lines ◽  
Single Agent ◽  
Myeloma Cell ◽  
Myeloma Cell Line

Abstract Introduction Multiple myeloma is a clonal plasma cell malignancy characterized by proliferation and accumulation of plasma cells in the bone marrow. Most patients are incurable with the current treatment modalities. Clearly novel agents are needed to improve the outcome for patients with myeloma. We have previously shown that the combination of bortezomib and tipifarnib results in synergistic myeloma cell death. This increase in apoptosis is associated with down regulation of phosphorylated AKT, a potent anti-apoptotic signaling molecule. Therefore, agents that target AKT represent ideal compounds for further study in myeloma. Perifosine is a novel, oral bioavailable alkylphospholipid. Perifosine has displayed apoptotic and antipropliferative activity in vitro and in vivo in several human cancer models including leukemia. Perifosine exerts its actions by interfering with key intracellular pathways including AKT, MAPK, JNK, p21waf1. Our hypothesis is that targeting AKT via multiple upstream pathways will result in increased myeloma cell apoptosis. Therefore, we assessed the effects of single agent perifosine with and without tipifarnib on multiple myeloma cell lines. Method The myeloma cell line RPMI8226 was used. Cell viability and proliferation were assessed using MTT assays. Cells were incubated with increasing concentrations of both agents alone and in combination. Cell proliferation was assayed at 24, 48 and 72 hours. Western blots were then carried out to evaluate the effects of the intracellular protein PDK1, one of the critical signaling molecules that phosphorylates and activates AKT. Results As we and others have previously shown, tipifarnib at concentrations that can be achieved clinically is associated with minimal cytotoxicity. At 5 μM, tipifarnib decrease proliferation by only 20%. In contrast, there is a potent dose response effect of single agent perifosine (Fig. 1). These results were apparent as early as 24 hours. When tipifarnib at 5 μM is used in combination with a subtherapeutic dose of perifosine (2 μM), there is a marked decrease in cell proliferation (Fig. 2). In addition, combination therapy resulted in a reduction in the phosphorylated form of PDK1, a critical finding that was not seen with either drug alone. Conclusion Combination therapy with tipifarnib and perifosine results in less cell proliferation compared to either agent used alone in the RPMI8226 myeloma cell line. The dosages employed in these in-vitro studies are lower than those used in previously published data and are clinically achievable. Studies targeting other cell lines including MM.1R, MM.1S, and U266 are in progress. Analysis of AKT, Caspase 3, 8 and 9 are being explored to help delineate the mechanism of this novel combination. The goal is to develop further effective treatment options for patients with myeloma. Figure 1 Figure 1. Figure 2 Figure 2.

scholarly journals CC-220 Is a Potent Cereblon Modulating Agent That Displays Anti-Proliferative, Pro-Apoptotic and Immunomodulatory Activity on Sensitive and Resistant Multiple Myeloma Cell Lines

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1591-1591 ◽  
Author(s):  
Chad C Bjorklund ◽  
Jian Kang ◽  
Ling Lu ◽  
Michael Amatangelo ◽  
Hsiling Chiu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Transcription Factors ◽  
Cell Line ◽  
Cell Lines ◽  
Clinical Investigation ◽  
Single Agent ◽  
Immunomodulatory Activity ◽  
Employment Equity ◽  
Equity Ownership

Abstract Background: CC-220 is a Cereblon (CRBN) binding compound currently under clinical investigation for systemic lupus erythematosus. Comparable to other Cereblon-binding agents, ex vivo treatment of CC-220 on B-cells, T-cells and monocytes leads to the degradation of the hematopoietic transcription factors Ikaros (IKZF1) and Aiolos (IKZF3).(1) Currently, CC-220 is being investigated in a phase Ib/IIa study CC-220-MM-001 (clintrial.gov trial #NCT02773030) as a single agent, or in combination with dexamethasone in relapsed/refractory multiple myeloma (RRMM) in patients who may have previously been exposed to pomalidomide. Here, we provide pre-clinical data and mechanistic rationale for the clinical development of CC-220 in heavily pre-treated RRMM. Results: In order to evaluate the ability of CC-220 effects on MM cells in vitro, we generated a large panel of MM cell lines (~69) that consist of 5 categories, including lenalidomide-sensitive (LS; n=26), intrinsically lenalidomide-resistant (ILR; n=7), acquired lenalidomide-resistant (ALR; n=12), acquired lenalidomide/dexamethasone-dual-resistant (ALDR; n=12), and acquired-pomalidomide-resistant (APR; n=12). Cell proliferation by 3H-thymidine incorporation at concentration between 0.01-100 μM was assessed by the area under the curve (AUC) for both CC-220 and pomalidomide. The average AUC was significantly reduced by 65% vs. 52% (p<0.01) for LS, 33% vs. 20% (p<0.01) for ILR, 30% vs. 20% (p<0.01) for ALR, 25% vs.10% (p<0.01) for ALDR, and 23% vs. 8% (ns) for PR cells for CC-220 vs. pomalidomide respectively. Apoptosis was analyzed by flow cytometry and AnnV+/ToPro3+ staining where CC-220 significantly (p<0.01) induced an average of 36% apoptotic cells compared to 30% for pomalidomide in LS cells, and 18% vs. 6% (p<0.5) in PS cells. Importantly, CC-220 showed anti-proliferative and pro-apoptotic activity in PR cells where Cereblon was still expressed. Additionally, both proliferation inhibition and apoptosis were synergistically enhanced across all cell line categories when CC-220 was used in combination with dexamethasone. We next evaluated the immunmodulatory effects on peripheral blood mononuclear cell (PBMCs)-stimulated killing of MM cells. Following a 72 hr incubation with CD3-stimulated PBMCs, CC-220 significantly induced the death of MM cells (~60%, across all cell type categories) within 4 hr, at concentrations more than 10-fold lower than pomalidomide. The observed CC-220-stimulated PBMC co-culture killing of MM cells closely correlated with dose-dependent increases in IL-2 secretion and Granzyme B release. Notably, CC-220 induced PBMC-mediated death of MM cells lacking observable Cereblon protein expression. Lastly, we evaluated the mechanism of action of CC-220 in MM cells in vitro. In the absence of Cereblon, as shown by shRNA knockdown or downregulation in a subset of PR cells, there is very little if any cell autonomous activity of CC-220, implicating Cereblon-dependency for its effects. Downstream of Cereblon, CC-220 stimulates the complete proteasomal degradation of both Ikaros and Aiolos in as little as 6 hr. Measurement of the half maximal time for 50% degradation of both Ikaros and Aiolos is kinetically faster from 1.9-2.9 vs. 2.4-6.9 hr depending on the MM cell line at a 10-fold lower dose for CC-220 compared to pomalidomide, respectively. CC-220 is also more efficient than pomalidomide at causing downregulation of the c-Myc/IRF4 axis, which has been shown to be essential for the cytotoxic effect of pomalidomide.(2) Conclusions: CC-220 is a potent anti-proliferative and pro-apoptotic compound that shows activity in several MM cell line categories with differing sensitivity to lenalidomide, pomalidomide and dexamethasone. Importantly, CC-220 induces PBMC-mediated killing of all MM cell lines regardless of the level of Cereblon expression and cell autonomous sensitivity. Mechanistically CC-220 acts through binding of Cereblon, leading to the degradation of the hematopoietic transcription factors Ikaros and Aiolos, followed by disruption of the MM promoting c-Myc/IRF4 axis. Taken together, these data support the clinical investigation of CC-220 in relapsed/refractory MM patients,who have previously been exposed to pomalidomide. Disclosures Bjorklund: Celgene Corporation: Employment, Equity Ownership. Kang:Celgene Corporation: Employment, Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Amatangelo:Celgene: Employment, Equity Ownership. Chiu:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership. Pourdehnad:Celgene Corporation: Employment, Equity Ownership. Klippel:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene: Employment, Equity Ownership.

scholarly journals Repurposing Leflunomide for Multiple Myeloma Treatment

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5667-5667
Author(s):  
Ralf Buettner ◽  
Corey Morales ◽  
Natalie Perret ◽  
Joycelynne Palmer ◽  
Amrita Krishnan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Plasma Cells ◽  
Single Agent ◽  
Differentially Expressed ◽  
Dependent Manner ◽  
Rpmi 8226

Abstract Background: Despite recent advances in treatment that have improved the prognosis for patients with multiple myeloma (MM), the disease remains incurable. There is a need for MM treatments with new mechanisms of action. Leflunomide, a commercially available oral immunosuppressive agent that has been FDA-approved since 1998 for the treatment of rheumatoid arthritis (RA) was evaluated as a potential MM therapy. The primary mechanism of action is de novo inhibition of pyrimidine synthesis by targeting dihydroorotate dehydrogenase (DHODH), and thus achieving an anti-proliferative effect in B- and T-lymphocytes. A secondary mechanism of action is inhibition of cytokine and growth factor receptor-associated tyrosine kinase activity. Methods and Results: Pre-clinical studies of teriflunomide, the active metabolite of leflunomide, showed that it inhibited cell growth and induced apoptosis in MM cell lines (MM.1S, MM.1R, U266, H929, RPMI-8226) and primary MM patients' (CD138+) plasma cells at clinically achievable concentrations (50-200 uM) in a time- and dose-dependent manner. We also found that teriflunomide induces cell-cycle arrest in both, glucocorticoid-sensitive (MM.1S) and resistant (MM.1R) MM cell lines at <200 uM. In addition, teriflunomide and dexamethasone synergized in the in vitro growth inhibition of MM cell line MM.1S. To identify MM-associated mRNAs and miRNAs whose expression levels are frequently altered upon teriflunomide exposure, MM cell lines (RPMI-8226, U266, MM.1S, NCI-H929) and CD138-enriched primary plasma cells from two MM patient samples were treated with 200 µM teriflunomide or DMSO control for 24 h before extraction and purification of mRNA and microRNA. mRNA-seq and miRNA-seq analysis from teriflunomide-treated MM samples revealed that similar changes were present between patient samples and cell lines. A total of 382 genes were found to be differentially expressed (225 upregulated, 157 downregulated). Upregulated genes included those that participate in defense response and negative regulation of cell growth. Genes involved in mitosis, rRNA biogenesis/processing, and immune response were generally downregulated. Analysis of microRNA-seq data from these samples revealed five differentially expressed, mostly newly discovered miRNAs that have unknown function. Conclusions: Leflunomide and its analogues demonstrated anti MM effects in vitro as well as synergy with dexamethasone. Based on our promising pre-clinical results we have initiated a single-agent phase I/II clinical trial in patients with relapsed/refractory MM. Disclosures No relevant conflicts of interest to declare.

Redox Proteins GSTs A1, M1, O1, O2, P1; Prx-3, and Trx-1 Association with In Vitro Imexon Sensitivity in Multiple Myeloma Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5077-5077
Author(s):  
Michael A. Thompson ◽  
Lan V. Pham ◽  
Linda Yoshimura ◽  
Amanda F. Baker ◽  
Evan M. Hersh ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Drug Exposure ◽  
Single Agent ◽  
Redox Proteins ◽  
Redox Protein ◽  
Future Studies ◽  
Protein Levels ◽  
Incurable Disease

Abstract Multiple myeloma (MM) is an incurable disease that is characterized by the proliferation of clonal plasma cells in the bone marrow. Conventional chemotherapy and autologous stem cell transplantation have improved survival. However, because MM remains an incurable disease, innovative approaches are needed. Imexon is a small-molecule, aziridine-containing iminopyrrolidone with previously demonstrated cytotoxic activity in MM. It acts by binding to sulfhydryls, decreasing glutathione levels, increasing reactive oxygen species (ROS), changing mitochrondial membrane potential, releasing cytochrome C into the cytoplasm, and activating caspases and thus inducing apoptosis. Phase I clinical trials have shown that imexon is relatively nontoxic and can induce responses in patients with diverse malignancies. Imexon has shown antitumor activity in vitro against a variety of tumors, with MM cell lines and MM fresh tumor cells having the highest sensitivity. Imexon is currently being studied in a phase I/II MM clinical trial. Since imexon depletes glutathione, we determined if the following basal levels of oxidation-reduction (redox) proteins would be predictive of sensitivity: glutathione S-transferases (GSTs) A1, M1, O1, O2, P1; peroxiredoxin-3 (Prx-3), and thioredoxin-1 (Trx-1). Imexon showed single agent activity against MM cell lines by XTT cell proliferation assays, consistent with previous reports (see Table). By Western blotting, the GSTs A1 and M1 were expressed at low levels, which did not seem to correlate with imexon sensitivity. GST P1 was expressed differentially in MM cell lines, but without an obvious correlation to imexon sensitivity. GST O1 and O2 showed modestly increased protein levels in the resistant 8226/IM10 cell line compared to the sensitive 8226/S cells. Prx-3 and Trx-1 were uniformly expressed except for the very low or absent expression of Trx-1 in MM-1 cells. While basal levels of most of the redox proteins tested were not predictive of imexon sensitivity, changes in redox protein levels after drug exposure may be predictive and will be tested in future studies. Additionally, other redox proteins may be better predictive markers. In conclusion, imexon is active as a single agent in vitro in MM cell lines. Basal levels of the redox proteins GST A1, M1, P1; Prx-3, and Trx-1 do not appear to correlate with imexon sensitivity in the cell lines tested. GST O1 and O2 are expressed at higher levels in the sensitive 8226/S cell line, suggesting potential utility as predictive markers. Future studies evaluating changes in redox protein levels after drug exposure in MM cell lines and in MM patients in an ongoing imexon clinical trial may further validate GST O1 and O2 as well as provide additional predictive molecular markers. Redox Protein Basal Levels in MM Cell Lines by Western Blot MM Cell Line (IC50, uM) GST A1 GST M1 GST O1 GST O2 GST P1 Prx-3 Trx-1 OCI (74) + + +++ +++ +++ +++ ++ 8226/S (95) − +/− ++ + + +++ ++ MM-1 (180) + +/− ++ + ++ +++ − 8226/IM10 (270) + +/− +++ ++ +/− +++ ++ U266/1300 (528) + +/− ++ + +++ +++ ++

PRL3 Is a Mediator of IL6/STAT3 Signaling and Defines a Population of Multiple Myeloma Patients Distinct from Those That Activate NFkB.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 671-671 ◽  
Author(s):  
Michael Sebag ◽  
Keith Stewart ◽  
Chang-Xin Shi ◽  
Yuan-Xiao Zhu ◽  
Wee-Joo Chng ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Plasma Cells ◽  
Luciferase Reporter ◽  
Mean Values ◽  
Stat3 Signaling ◽  
First Intron

Abstract Previous work in our laboratory has identified the importance of the NFB signaling pathway for plasma cell (PC) survival and accumulation in Multiple Myeloma (MM). Although up to 43% of patient PCs appear to have activated NFkB by gene expression profiling (GEP), the remainder must rely on an alternate signaling pathway for their survival. We examined our MM GEP datasets and supervised the clustering of patients according to an index of NFkB activity. The majority of patients not activating NFkB were associated with a high IL-6/Stat3 signature, which included the protein phosphatase of regenerating liver-3 (PRL-3). Interestingly, analysis of the PRL-3 promoter revealed competing binding sites for both NFkB and STAT3 within the first intron, suggesting that these play a role in the transcriptional regulation of this gene. PRL-3 has been previously demonstrated to be absent in most normal tissues while its expression in MM cells is quite elevated. As normal PCs have low levels of PRL-3 expression, we determined the upper limit of normal to be 3SD above the mean of these cells. Using this cutoff we report that elevated PRL-3 expression is observed in plasma cells from 1/15 normal individuals, 4/22 MGUS patients and 47/126 patients with either smoldering or overt MM (6.7%, 18.2% and 45.6% respectively). Less than 10% of patients were seen to have both an elevated NFkB index and high PRL-3 expression. Patients with higher expression of PRL-3 were found to have statistically significant higher mean values of creatinine, C-reactive protein, lower albumin levels and a lower overall survival in our Mayo Clinic MM dataset. To examine the role PRL-3 in MM biology we first stimulated MM cell lines with IL-6 and observed PRL-3 up-regulation in two cell lines (INA6, OPM1) but not in JK6L (an NFkB activated cell line). Using a luciferase reporter we have determined that STAT3 induction of PRL3 expression requires the first intron of the PRL3 gene which contains the putative STAT3 binding site. Finally we show that PRL-3 inhibition by shRNA leads to decreased cell growth and loss of IL-6 responsiveness in vitro in at least one cell line. Based on these results, we propose that the majority of MM patients depend on either the NFkB or STAT3 signaling pathways for their survival and that these are to a large degree mutually exclusive. Our experimental results suggest that PRL-3 plays a role in IL-6/STAT3 signaling and that it is a promising target for drug development.

Ruxolitinib Regulates the Autophagy Machinery in Multiple Myeloma Cells

2020 ◽  
Vol 20 (18) ◽  
pp. 2316-2323 ◽  
Author(s):  
Alican Kusoglu ◽  
Bakiye G. Bagca ◽  
Neslihan P.O. Ay ◽  
Guray Saydam ◽  
Cigir B. Avci
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
B Lymphocyte ◽  
Plasma Cells ◽  
Annexin V ◽  
Myeloma Cell ◽  
Control Group ◽  
Multiple Myeloma Cell ◽  
Ic50 Values ◽  
Stat Pathway

Background: Ruxolitinib is a selective JAK1/2 inhibitor approved by the FDA for myelofibrosis in 2014 and nowadays, comprehensive investigations on the potential of the agent as a targeted therapy for haematological malignancies are on the rise. In multiple myeloma which is a cancer of plasma cells, the Interleukin- 6/JAK/STAT pathway is emerging as a therapeutic target since the overactivation of the pathway is associated with poor prognosis. Objective: In this study, our purpose was to discover the potential anticancer effects of ruxolitinib in ARH-77 multiple myeloma cell line compared to NCI-BL 2171 human healthy B lymphocyte cell line. Methods: Cytotoxic effects of ruxolitinib in ARH-77 and NCI-BL 2171 cells were determined via WST-1 assay. The autophagy mechanism induced by ruxolitinib measured by detecting autophagosome formation was investigated. Apoptotic effects of ruxolitinib were analyzed with Annexin V-FITC Detection Kit and flow cytometry. We performed RT-qPCR to demonstrate the expression changes of the genes in the IL-6/JAK/STAT pathway in ARH-77 and NCI-BL 2171 cells treated with ruxolitinib. Results: We identified the IC50 values of ruxolitinib for ARH-77 and NCI-BL 2171 as 20.03 and 33.9μM at the 72nd hour, respectively. We showed that ruxolitinib induced autophagosome accumulation by 3.45 and 1.70 folds in ARH-77 and NCI-BL 2171 cells compared to the control group, respectively. Treatment with ruxolitinib decreased the expressions of IL-6, IL-18, JAK2, TYK2, and AKT genes, which play significant roles in MM pathogenesis. Conclusion: All in all, ruxolitinib is a promising agent for the regulation of the IL-6/JAK/STAT pathway and interferes with the autophagy mechanism in MM.

scholarly journals Global Genomic Analysis of Newly Diagnosed t(4 ;14) Multiple Myeloma Reveals a Specific Mutational Spectrum and Identifies PKD2 As a Potential Therapeutic Target

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4462-4462
Author(s):  
Xiu Ly Song ◽  
Raphaël Szalat ◽  
Alexis Talbot ◽  
HaiVu Nguyen ◽  
Mehmet K. Samur ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Plasma Cells ◽  
Genomic Analysis ◽  
Sequencing Analysis ◽  
Newly Diagnosed ◽  
Potential Therapeutic Target ◽  
Mutational Landscape

Abstract In Multiple Myeloma (MM), the t(4;14) translocation is associated with a poor outcome. However, beside this translocation, the genetic events which determine the adverse evolution of the disease and the resistance to treatments remain elusive. In this study we performed whole exome or RNA sequencing analysis of samples from 65 newly diagnosed t(4;14) MM. We found that NRAS, KRAS, MAPK and FGFR3 are frequently mutated (12%, 9%, 13.8%, and 20% respectively). Overall, the FGFR3/RAS/BRAF/MAPK genes were mutated in 36 cases (54%). There was a negative correlation between mutations in FGFR3 and those occurring in NRAS, KRAS and BRAF as expected from the mutually exclusive occurrence of mutations in these genes. In addition to alterations in TP53 and DIS3, we found marked elevated frequency of mutations in PRKD2 (10.7%), ATM/ATR (10.7%) and MYCBP2 (7.6%), reduced frequency in FAM46C (1.5%) and no mutation in TRAF3 and CCND1. Mutations in ATM/ATR were strongly associated with the MB4-2 breakpoint (Bp) (p = 1.62 10-4) and significantly correlated with mutations affecting genes coding for members of the MAPK family. We observed a positive correlation between non-silent mutations in PRKD2 and the MB4-1 or MB4-3 Bp (p = 1.3 10-2). Of note, PRKD2 mutations are exclusively found in 3 t(4;14) MM cell lines and among the 84 MM sequenced by Bolli et al. (1), none of the non t(4;14) patient were mutated in PRKD2, indicating that this genetic lesion is associated with t(4;14) MM. In the NCI-H929 t(4;14) MM cell line, which is mutated for PRKD2, encoding the PKD2 serine/threonine kinase, we observed elevated levels of phosphorylated PKD2. Furthermore, inhibition of PKD, decreased PKD2 phosphorylation and triggered reduced proliferation and apoptosis of MM cell lines and fresh plasma cells from patients in vitro. These results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients. Altogether, these results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients. Reference 1. Bolli, N., Avet-Loiseau, H., Wedge, D.C., Van Loo, P., Alexandrov, L.B., Martincorena, I., Dawson, K.J., Iorio, F., Nik-Zainal, S., Bignell, G.R., et al. (2014). Heterogeneity of genomic evolution and mutational profiles in multiple myeloma. Nat Commun 5, 2997. Disclosures Munshi: Janssen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Patents & Royalties.

A Novel Pan-PI3K/Akt Inhibitor, SF1126, Inhibits In Vitro Growth of Multiple Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4806-4806
Author(s):  
Jeannine Silberman ◽  
Kimberly Dalbey ◽  
Claire Torre ◽  
Ebenezer David ◽  
Leif Bergsagel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Growth Inhibition ◽  
Cell Line ◽  
Cell Lines ◽  
Mtt Assay ◽  
Immunoblot Analysis ◽  
Akt Inhibitor ◽  
Downstream Targets

Abstract Backround: Dysregulation of the PI3K/Akt signal transduction pathway has been implicated in the development of a number of malignancies, including multiple myeloma (MM). This cellular signaling mechanism and its downstream targets (eg mTOR) regulate cell growth, proliferation and apoptosis. SF1126 (Semafore) is a water soluble prodrug of the pan-PI3K inhibitor, LY294002, whose anti-proliferative and pro-apoptotic activity has been well described in the literature. Preclinical studies using SF1126 in a variety of malignancies including glioma, prostate, non-small cell lung cancer, and breast cancer appear promising and have demonstrated profound antiangiogenic effects mediated through VEGF inhibition. Aim: To demonstrate in vitro anti-myeloma activity of SF1126, alone and in combination with dexamethasone, bortezomib, and melphalan and evaluate their effects on downstream targets of PI3K/Akt. Methods: MM cell lines (MM.1R, MM.1S, RPMI 8226) were treated with SF1126 (1–100uM), dexamethasone (5uM), bortezomib (5nM), melphalan (10uM) alone, and in combination. Growth inhibition following treatment was measured by MTT assay at 24 and 48 hours. Apoptosis was assessed by annexin-V binding assay using flow cytometry. Immunoblot analysis was performed to measure downstream targets of Akt including: p-PDK1 and mTOR (4E-BP1). Results: A clear dose response was established with an IC50 of 8.75uM in the MM.1R and 7.5uM in the MM.1S cell lines at 48 hours. At 24 and 48 hours, 5uM SF1126 alone resulted in 80% and 64% cell viability by MTT assay, respectively, in the MM.1R cell line. The combination of 5uM SF1126 with conventional agents was then tested in the MM.1R cell line. Combination with 5uM dexamethasone enhanced the efficacy of 5uM SF1126 by 26% at 48 hours. Combination with 10uM melphalan enhanced the efficacy of 5uM SF1126 by 20% at 24 hours. The combination with 5nM bortezomib enhanced the efficacy of 5uM SF1126 by 23% at 48 hours. Given prior experience demonstrating that short exposure to bortezomib activates Akt, we tested sequential administration of bortezomib and SF1126 in the MM.1R cell line. Optimal cell death was induced with bortezomib prior to SF1126, followed by concurrent administration. Immunoblot analysis of p-PDK1, downstream mTOR target (4E-BP1) were performed on the MM.1S cell line treated with 5, 10, 20, and 50uM SF1126 at 12 and 24 hours. At the 12 hour time point, p-PDK-1 appeared to increase, but was significantly reduced by 48 hours. A similar pattern of initial upregulation followed by reduction by 24 hours was seen with the mTOR protein 4E-BP1. Conclusion: SF1126 has dose dependent, in vitro activity in several multiple myeloma cell lines both as a single agent and in combination with dexamethasone, bortezomib, and melphalan. The addition of SF1126 to dexamethasone in a dexamethasone resistant cell line results in increased cell death, possibly by overcoming resistance mechanisms. The addition of SF1126 to bortezomib and melphalan also resulted in increased growth inhibition over either agent alone. These results warrant further study of this promising new pan-PI3K/Akt inhibitor.

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