Inhibition of Wnt/ß-Catenin Pathway with ICG-001 Induces Apoptosis in Multiple Myeloma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2951-2951
Author(s):  
Eileen R Grigson ◽  
Alexandra Pisklakova ◽  
Brittany Weissman ◽  
Yulia Nefedova
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Wnt Signaling ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Caspase 3 ◽  
Control Group ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt ◽  
Rpmi 8226

Abstract Abstract 2951 Multiple Myeloma (MM) is characterized by clonal expansion of malignant plasma cells, which preferentially reside in the bone marrow (BM). It has been previously shown that canonical Wnt signaling plays a critical role in proliferation and differentiation in MM. ICG-001 is a selective Wnt/ß-catenin signaling inhibitor that binds to the amino terminus of CREB, thus preventing ß-catenin/TCF signaling in the nucleus. Here we investigated whether the pharmacological inhibition of canonical Wnt signaling with ICG-001 could affect viability of MM cells and produce anti-myeloma effect. C-myc and cyclin D1 are known to be downstream target genes of canonical Wnt signaling. MM cells lines treated with ICG-001 revealed down regulation of c-myc and cyclin D1 as compared to control. To investigate the varying sensitivity of cell lines to ICG-001, four cells lines were used: RPMI-8226, NCI-H929, U266, and MM1S. MM cells lines were treated with ICG-001 (1uM – 40uM) and data was collected using MTT assay or flow cytometry (Annexin binding assay). ICG-001 significantly reduced viability in all four cell lines due to induction of apoptosis, with RPMI-8226 showing the greatest sensitivity. We performed cleaved-caspase assays and demonstrated that apoptosis was caspase-3 dependent. We have also evaluated the effect of ICG-001 in BM samples from patients with MM. BM cells were treated with ICG-001 for 24 hours and the level of cleaved-caspase-3 was evaluated by flow cytometry. MM cells were defined as double positive CD138+ and Lambda/Kappa+. Our results demonstrated that MM cells were sensitive to ICG-001, while this drug was not able to induce cell death in non-tumor BM cells. To test the effect of ICG-001 in vivo we injected SCID/Beige mice with 10 × 105 8226 cells in 100 μL phosphate-buffered saline (PBS). After 3 weeks when a tumor was measurable, the mice were split into two groups and were treated with either ICG-001 (100 mg/kg) or vehicle control (PBS) daily, for 14 days. Tumor size was consistently monitored during treatment and 3 weeks after treatment. We observed that ICG-001 produced a significant antitumor effect as compared to control group. These data indicate targeting Wnt/ß-catenin Pathway with ICG-001 could be therapeutically beneficial to patients with MM. Disclosures: No relevant conflicts of interest to declare.

Bortezomib Induces Activation of b-Catenin/TCF Signaling Pathway in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1851-1851
Author(s):  
Ya-Wei Qiang ◽  
Bo Hu ◽  
Yu Chen ◽  
Wei Qiang ◽  
Christoph Heuck ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Transcriptional Activity ◽  
Luciferase Reporter ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt ◽  
Dose Dependent

Abstract Abstract 1851 Background: The proteasome inhibitor Bortezomib (Bz) shows significant activity in Multiple Myeloma (MM) by acting on MM cell directly as well as by augmenting bone formation in vitro and in vivo. Its effect on the bone could be traced to promoting differentiation of mesenchymal stem cells into osteoblast cells by regulating BMP2 and canonical Wnt signaling. However, the molecular mechanism mediating the direct anti-MM activity of Bz remains to be fully understood. Initially the rationale for the use of Bz in MM was inhibition of NF-kB signaling, yet subsequent studies showed that Bz actually induces activation of this pathway. In this study, we examined whether Bz regulates the activity of canonical Wnt signaling pathway in MM and whether the growth-inhibition effect of Bz was associated with activation of this pathway by using multiple MM cell lines including EJM, H929, INA6, KMS28BM, JJN3, L363, OPM1, OPM2, RPMI8226, UTMC, XG2 and XG6 as well as primary plasma cells (PC) from six patients with newly diagnosed MM. Methods/Results: Immunoblotting demonstrated that Bz induces stabilization of b-catenin protein in three MM cell lines (H929, OPM2 and UTMC) in a time- and dose-dependent manner. These changes were not seen when the same cell lysate were immunoblotted for other catenin family members, a-catenin and g-catenin. Increased levels of b-catenin protein response to Bz treatment were observed in other 9 MM cell lines (EJM, INA6, KMS28BM, JJN3, L363, OPM1, RPMI8226, XG2 and XG6) and in the 6 CD138+ sorted bone marrow PC from patients with MM. To determine if Bz regulation of b-catenin level is a specific effect of the inhibition of 26S proteasome subunit we treated the same MM cell lines with another proteasome inhibitor, MG132. Similar results were observed in response to MG132 for all four MM cell lines, suggesting the effect of Bz on b-catenin protein is 26S proteosome inhibitor specific. Increases in b-catenin protein levels in MM cells were not due to increased Ctnnb1/CTNNB (b-catenin) gene transcription as b-catenin mRNA did not change in these cells treated with Bz. These results indicate that proteasome inhibition increases b-catenin is independent of transcriptional upregulation. To determine whether Bz induces the nuclear localization and transcriptional activity of b-catenin, cells were incubated with Bz for 6 hours and then fractionated to separate the nuclear and cytoplasmic fractions. Treatment with Bz resulted an increase in nuclear b-catenin as well as b-catenin in cytoplasm in four cell lines including H929, INA6, OPM1 and MM144. Increase in cytoplasmic and nuclear b-catenin was further confirmed by immunofluorescence with antibodies specific for active form of b-catenin. To determine whether Bz affects b-catenin-mediated transcriptional activity, we used a TCF/LEF luciferase reporter construct cloned in lentiviral vector. OPM2 cells were infected with lentiviral particle containing the TCF reporter or containing empty vector and were then treated with serial concentrations of Bz. The luciferase activity exhibited a dose-dependent response to Bz analogous to the stabilization of b-catenin. Similar results were observed in 7 out of 8 MM cell lines compared with untreated control. Stimulation of TCF transcriptional activity by Bz was independent of modifiers of extracellular Wnt ligands, such as Frizzled receptors, LRP5/6 co-receptors and sFRPs or the activation of intracellular GSK3b. Conclusion: These results indicate that Bz augments activation of canonical Wnt signaling by preventing b-catenin protein from proteosome-mediated degradation in MM cells. Concentrations of Bz for stimulating TCF transcriptional activity are comparable to those being used to induce inhibition of MM proliferation. Experiments modulating cytoplasmic as well as the nuclear players and interactions of the Wnt-pathway are ongoing to determine if Bz mediated activation of b-catenin signaling is responsible for its direct anti-MM effect. Disclosures: Barlogie: Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

Nuclear β-catenin localization is not sufficient for canonical Wnt signaling activation in human melanoma cell lines

2011 ◽  
Vol 45 (5) ◽  
pp. 816-822 ◽  
Author(s):  
K. V. Kulikova ◽  
A. V. Posvyatenko ◽  
N. V. Gnuchev ◽  
G. P. Georgiev ◽  
A. V. Kibardin ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Human Melanoma ◽  
Human Melanoma Cell ◽  
Canonical Wnt Signaling ◽  
Signaling Activation ◽  
Canonical Wnt ◽  
Melanoma Cell Lines

scholarly journals Abi1 loss drives prostate tumorigenesis through activation of EMT and non-canonical WNT signaling

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Disharee Nath ◽  
Xiang Li ◽  
Claudia Mondragon ◽  
Dawn Post ◽  
Ming Chen ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Wnt Signaling ◽  
Tumor Progression ◽  
Cell Line ◽  
Rna Sequencing ◽  
Cell Lines ◽  
Adaptor Protein ◽  
Prostate Tumor ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt

Abstract Background Prostate cancer development involves various mechanisms, which are poorly understood but pointing to epithelial mesenchymal transition (EMT) as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, acts as tumor suppressor in prostate cancer but the role of ABI1 in EMT is not clear. Methods To investigate the molecular mechanism by which loss of ABI1 contributes to tumor progression, we disrupted the ABI1 gene in the benign prostate epithelial RWPE-1 cell line and determined its phenotype. Levels of ABI1 expression in prostate organoid tumor cell lines was evaluated by Western blotting and RNA sequencing. ABI1 expression and its association with prostate tumor grade was evaluated in a TMA cohort of 505 patients and metastatic cell lines. Results Low ABI1 expression is associated with biochemical recurrence, metastasis and death (p = 0.038). Moreover, ABI1 expression was significantly decreased in Gleason pattern 5 vs. pattern 4 (p = 0.0025) and 3 (p = 0.0012), indicating an association between low ABI1 expression and highly invasive prostate tumors. Disruption of ABI1 gene in RWPE-1 cell line resulted in gain of an invasive phenotype, which was characterized by a loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 spheroids. Through RNA sequencing and protein expression analysis, we discovered that ABI1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of ABI1. Furthermore, an increase in STAT3 phosphorylation upon ABI1 inactivation and the evidence of a high-affinity interaction between the FYN SH2 domain and ABI1 pY421 support a model in which ABI1 acts as a gatekeeper of non-canonical WNT-EMT pathway activation downstream of the FZD2 receptor. Conclusions ABI1 controls prostate tumor progression and epithelial plasticity through regulation of EMT-WNT pathway. Here we discovered that ABI1 inhibits EMT through suppressing FYN-STAT3 activation downstream from non-canonical WNT signaling thus providing a novel mechanism of prostate tumor suppression.

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.

Beta-Catenin Depended and Independent Effects Induced by Myeloma Cells in Human and Murine Osteoblasts and Osteoblast Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3433-3433
Author(s):  
Francesca Morandi ◽  
Sara Tagliaferri ◽  
Sabrina Bonomini ◽  
Mirca Lazzaretti ◽  
Luca Ferrari ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Cell Lines ◽  
Osteoblastic Cells ◽  
Beta Catenin ◽  
Canonical Wnt Signaling ◽  
Osteoprogenitor Cell ◽  
Significant Difference ◽  
Bone Biopsies ◽  
Canonical Wnt ◽  
Osteoblast Progenitors

Abstract Osteoblast impairment occurs within myeloma (MM) cell infiltration into the bone marrow (BM). Wnt signaling is involved in the regulation of osteoblast formation. Canonical Wnt signaling pathway is activated by Wnt 1/3a that induce the activation of GSK3/Axin complex leading to the stabilization and nuclear translocation of beta-catenin that in turn activates the transcription system Lef1/TCF. Recently it has been reported that MM cells produce the Wnt inhibitors DKK-1 demonstrating a correlation between its expression and the presence of bone lesions in MM patients. However the effect of MM cells on Wnt signaling cascade in osteoblasts and osteoblast progenitors has not been investigated. To clarify this issue, first we checked DKK-1 production by human myeloma cell lines (HMCLs), purified CD138+ MM cells and BM plasma of MM patients by PCR and ELISA. Following we performed a co-culture system with HMCLs or CD138+ MM cells and either human osteoblast line (HOBIT) and with BM osteoprogenitor cells (PreOB) obtained after differentiation from mesenchymal cells or murine osteoprogenitor cell lines C2C12 and MC3T3. Both DKK-1 positive HMCLs (XG-1 and JJN3) and negative ones (RPMI-8226, OPM-2) have been used in co-culture as well as DKK-1 positive and negative purified CD138+ MM cells. Similarly we tested the effect of BM plasma of MM patients positive and negative for DKK-1 production on both human and murine cells. Wnt signaling in osteoblasts and osteoblast progenitors was evaluated either at mRNA level by specific human and murine Wnt Array kits and by quantitative PCR or at protein one by Western blot analysis for GSK3b/Axin and LEF-1/TCF expression. We evaluated active de-phosphorylated beta-catenin and inactive phosphorilated one by westernblot and by ELISA in cytosolic and nuclear extracts. DKK-1 median levels detected in the conditioned media of XG-1 and JJN3, MM cells and in BM plasma of DKK-1 positve MM patients were 0.60 ng/mL and 0.38 and 8.84 (range: 1.55–91) ng/mL respectively. Any significant inhibitory effect on WNT signaling and active beta-catenin expression and levels was not observed in HOBIT and human PreOB after co-culture with both HMCLs and MM cells or BM plasma independently to DKK-1 expression. On the contrary DKK-1 positive MM cells or BM plasma suppressed active beta-catenin expression in murine osteoprogenitor cell lines in presence of BMP-2. Consistently Wnt3a stimulation as well as anti-DKK-1 abs. did not restore the inhibitory effects on osteoblast formation and differentiation induced by MM cells in human PreOB. Consistently any significant difference was not detected on beta-catenin expression by stromal/osteoblastic cells on bone biopsies by immunohistochemistry between osteolytic (n°=10) and non-osteolytic (N°=10) MM patients. The different behavior between human and murine osteoblastic cells was further investigated. We found that both cells expressed significant levels of active beta-catenin however DKK-1 suppressed active nuclear and cytosol beta-catenin at concentration of 20–30 ng/mL in C2C12 and MC3T3 whereas only DKK-1 concentrations higher to 500 ng/mL are able to inhibited beta-catenin in HOBIT and human PreOB as well as osteoblast formation and differentiation in human BM cultures. In conclusion our data indicate that MM cells block canonical Wnt signaling in murine osteoblastic cells but not in human osteoblasts and osteoblast progenitors. Beta-catenin independent mechanisms could be involved in DKK-1 mediated bone destruction in MM patients.

AML Survival and In Vivo Progression Is Dependent On β-Catenin Signaling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2398-2398
Author(s):  
Elena K Siapati ◽  
Magda Papadaki ◽  
Zoi Kozaou ◽  
Erasmia Rouka ◽  
Evridiki Michali ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Wnt Signaling Pathway ◽  
Canonical Wnt Signaling ◽  
Down Regulation ◽  
Canonical Wnt

Abstract Abstract 2398 Poster Board II-375 B-catenin is the central effector molecule of the canonical wnt signaling pathway which governs cell fate and differentiation during embryogenesis as well as self-renewal of hematopoietic stem cells. Deregulation of the pathway has been observed in various malignancies including myeloid leukemias where over-expression of β-catenin is an independent adverse prognostic factor. In the present study we examined the functional outcome of stable β-catenin down-regulation through lentivirus-mediated expression of short hairpin RNA (shRNA). Reduction of the β-catenin levels in AML cell lines and patient samples diminished their in vitro proliferation ability without significantly affecting cell viability. In order to study the role of β-catenin in vivo, we transplanted leukemic cell lines with control or reduced levels of β-catenin in NOD/SCID animals and analyzed the engraftment levels in the bone marrow. We observed that while the immediate homing of the cells was not affected by the β-catenin levels, the bone marrow engraftment was directly dependent on its levels. Subsequent examination of bone marrow sections revealed that the reduced engraftment was partly due to the inability of the cells with lower β-catenin levels to dock to the endosteal niches, a finding that was confirmed in competitive repopulation assays with untransduced cells. When we examined the expression levels of adhesion molecules and integrins in engrafted cells in vivo, we observed a significant down-regulation of CD44 expression, a molecule that participates in the interaction of HSCs with the niche. Gene expression analysis of the components of the wnt signaling pathway showed that the pathway is subject to tight transcriptional regulation with minor expression deviations. We did, however, observe an up-regulation in components that participate in the non-canonical wnt signaling pathways such as the WNT5B ligand. Ongoing experiments in normal cord blood CD34+ cells will determine the in vivo role of β-catenin signaling in normal hematopoietic progenitors. In conclusion, our study showed that β-catenin comprises an integral part in the development and progression of AML in vivo, indicating that manipulation of the wnt pathway may hold a therapeutic potential in the management of AML. Disclosures: No relevant conflicts of interest to declare.

Anti-Tumor Activity of Novel Small Molecule Wnt Signaling Inhibitor, CWP232291, In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3038-3038 ◽  
Author(s):  
Joo Young Cha ◽  
Ji-Eun Jung ◽  
Kwan-Hoo Lee ◽  
Isabelle Briaud ◽  
Fnu Tenzin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Wnt Signaling ◽  
Caspase 3 ◽  
Target Genes ◽  
Plasma Cells ◽  
Wnt Signaling Pathway ◽  
Beta Catenin ◽  
Tumor Bearing ◽  
Induction Of Apoptosis ◽  
Rpmi 8226

Abstract Abstract 3038 Multiple myeloma (MM), one of the most incurable hematological malignancies in adults, is a disorder of plasma cells characterized by accumulation of clonal proliferation of malignant plasma cells in the bone marrow (BM). Overexpression of beta-catenin, the downstream effector of the canonical Wnt signaling pathway, has been reported in both MM cell lines and patient samples. Activated Wnt signaling pathway has also been reported to play a critical role in progression of MM cell proliferation, thus highlighting the need for new therapeutic approaches, particularly those targeting Wnt molecular pathway. Here we report the discovery of a novel inhibitor of Wnt signaling CWP232291, which promotes degradation of beta-catenin. CWP232291 exhibits potent growth inhibitory activity in several MM cell lines (RPMI-8226, OPM-2, NCI-H929, JJN3, and EJM) with IC50 values of 13 – 73 nM. The inhibitory activity of CWP232291 on Wnt signaling is demonstrated by reporter gene assay and by its effect in down-regulation of Wnt target genes. Using HEK293 cells, CWP232291 treatment dose dependently reduces promoter activity of TOPflash induced by Wnt-3a-Conditioned Media, at a calculated IC50 value of 273 nM. Furthermore, MM cells treated with CWP232291 show downregulated expression of Wnt target genes such as survivin by triggering degradation of beta-catenin. Treatment of these cells with CWP232291 results in activation of caspase-3 and PARP cleavage, indicating induction of apoptosis. To investigate the potential in vivo anti-tumor efficacy of CWP232291, we utilized two MM tumor bearing mice models. Daily or intermittent intravenous (i.v.) administration of CWP232291 led to significant tumor growth inhibition (TGI) in OPM-2 (50 mg/kg, qdx5: regression and 100 mg/kg, biw: 95% TGI) and RPMI-8226 (100 mg/kg, qdx5: regression and 100 mg/kg tiw: 80% TGI) xenograft model with no obvious change in body weight. The anti-tumor efficacies of CWP232291 were dose-dependent and had strong correlations with degradation of beta-catenin in the tumor samples. Potent induction of apoptosis through cleavage of Caspase-3 and PARP and significant decrease of plasma M protein levels in OPM-2 tumor bearing mice were detected as early as 2 and up to 24 hours after single i.v. administration of CWP232291. Taken together, these data clearly demonstrate the impressive anti-tumor profile of CWP232291 with a good therapeutic window and suggest a potential therapeutic application of CWP232291 for the treatment of MM. Disclosures: Cha: Choongwae Pharma Corp.: Employment. Jung:Choongwae Pharma Corp.: Employment. Lee:Choongwae Pharma Corp.: Employment. Briaud:Theriac Pharmaceutical Corp.: Employment. Tenzin:Theriac Pharmaceutical Corp.: Employment. Jung:Choongwae Pharma Corp.: Employment. Pyon:Choongwae Pharma Corp.: Employment. Lee:Choongwae Pharma Corp.: Employment. Chung:Choongwae Pharma Corp.: Employment. Lee:Choongwae Pharma Corp.: Employment. Oh:Choongwae Pharma Corp.: Employment. Jung:Choongwae Pharma Corp.: Employment. Pai:Choongwae Pharma Corp.: Employment. Emami:Theriac Pharmaceutical Corp.: Employment.

SNS01-T Exhibits Significant Anti-Tumoral Activity In Models Of Multiple Myeloma and Non-Hodgkins B Cell Lymphoma and Induces Cell Death In Malignant But Not Normal B Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 880-880
Author(s):  
Catherine A Taylor ◽  
Terence Tang ◽  
Sarah Francis ◽  
Zhongda Liu ◽  
Qifa Zheng ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Tumor Volume ◽  
Cell Lymphoma ◽  
Xenograft Model ◽  
B Cell Lymphoma ◽  
Control Group ◽  
Rpmi 8226

Abstract SNS01-T is a novel nanoparticle that is designed to selectively initiate apoptosis in B-cell cancers such as multiple myeloma and non-Hodgkins B-cell lymphomas. SNS01-T comprises a plasmid DNA (pExp5A) encoding a pro-apoptotic form of the eukaryotic translation initiation factor 5A (eIF5A) containing a single-point mutation that prevents hypusination, an eIF5A siRNA that inhibits expression of the pro-survival hypusine-eIF5A protein, and a polymer that serves to assemble the nucleic acids into a nanoparticle. SNS01-T is currently being investigated in a multi-site, open-label Phase1b/2a dose escalation study in subjects with relapsed or refractory multiple myeloma (MM), mantle cell lymphoma (MCL), or diffuse large B cell lymphoma (DLBCL). SNS01-T has demonstrated activity in MM xenograft models as well as in B cell lymphoma models of MCL and DLBCL, when administered twice weekly at doses ≥ 0.18 mg(nucleic acid)/kg. In this study we compared the ability of SNS01-T to transfect, regulate eIF5A expression, and kill MM, DLBCL, and MCL cell lines. Furthermore, the activity of SNS01-T in normal B cells was investigated. A previous study using a KAS-6/1 MM xenograft model demonstrated that the eIF5A siRNA and plasmid pExp5A both have anti-tumoral activity in MM but had a greater impact on tumour growth when combined together as SNS01-T. This finding was confirmed in this study in a second MM model (RPMI 8226) as well as in a DLBCL xenograft model. To determine the efficiency of SNS01-T transfection into malignant or normal B cells, the pExp5A plasmid and eIF5A siRNA were labeled with FITC and DY547, respectively, packaged into nanoparticles using polyethylenimine polymer, and used to transfect cultured cells. FACS analysis was used to determine the percent of the cell population transfected with plasmid, siRNA, or both. RT-qPCR was used to assess biological activity of SNS01-T by quantifying the expression of eIF5AK50R mRNA transgene and endogenous eIF5A mRNA in a variety of B cell lines. The IC50 of SNS01-T in a panel of MM, MCL, and DLBCL cell lines was determined by XTT assay. SCID mice bearing either RPMI 8226 MM tumours or SuDHL6 GCB DLBCL tumours were treated with pExp5A plasmid (formulated with PEI and control siRNA), eIF5A siRNA (formulated with PEI and a control plasmid), or SNS01-T at 0.375 mg/kg twice per week by intravenous injection. SNS01-T was able to transfect MM, MCL, and DLBCL cell lines, although the proportion of cells transfected with both plasmid and siRNA was higher in MM cells. Transfection of SNS01-T resulted in expression of the transgene as well as a statistically significant reduction in expression of eIF5A mRNA compared to untreated controls for all three cell types. In contrast, normal B cells were found to take up fluorescently-labeled SNS01-T with reduced efficiency compared to RPMI 8226 MM cells. Futhermore, SNS01-T was observed to induce cell death in RPMI 8226 MM cells but not in normal B cells. In the RPMI 8226 xenograft model, treatment with either the pExp5A plasmid alone or eIF5A siRNA alone resulted in a 66 % reduction (p < 0.0001) or 44 % reduction (p < 0.05) in tumor volume compared to the control group at day 24 of the study. In contrast, treatment with SNS01-T, which contains both the pExp5A plasmid and the eIF5A siRNA, resulted in an 86 % (p < 0.0001) reduction in tumor volume. A similar result was observed in the SuDHL6 model with a 14 % reduction or 27 % reduction (p < 0.05) in tumor volume compared to the control group at day 20 of the study following treatment with pExp5A plasmid or eIF5A siRNA, respectively. In contrast, treatment with SNS01-T resulted in a 79 % (p < 0.0001) reduction in tumor volume. Collectively, these preclinical studies indicate that SNS01-T therapy has significant potential against MM, MCL, and DLBCL. Disclosures: Taylor: Senesco Technologies: stock options Other. Dondero:Senesco Technologies: Employment. Thompson:Senesco Technologies: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

scholarly journals Differentiation-Inducing Factor-1 Alters Canonical Wnt Signaling and Suppresses Alkaline Phosphatase Expression in Osteoblast-Like Cell Lines

2006 ◽  
Vol 21 (8) ◽  
pp. 1307-1316 ◽  
Author(s):  
Etsuko Matsuzaki ◽  
Fumi Takahashi-Yanaga ◽  
Yoshikazu Miwa ◽  
Masato Hirata ◽  
Yutaka Watanabe ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Wnt Signaling ◽  
Cell Lines ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt ◽  
Alkaline Phosphatase Expression

scholarly journals Tris DBA Palladium Overcomes Hypoxia Mediated Drug Resistance in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2978-2978
Author(s):  
Pilar De La Puente ◽  
Barbara Muz ◽  
Feda Azab ◽  
Micah John Luderer ◽  
Jack L. Arbiser ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Drug Resistance ◽  
Cell Lines ◽  
Research Funding ◽  
Caspase 3 ◽  
Cellular Processes ◽  
Hypoxic Conditions

Abstract Introduction: Despite recent progress in novel and targeted therapies, multiple myeloma (MM) remains a therapeutically challenging incurable disease. The regulation of important cellular processes and its link to cancer presented Src as an attractive target for MM. Src is a non-receptor protein tyrosine kinase which regulates multiple fundamental cellular processes including cell growth, migration, survival and differentiation. Activated Src in cancer lead to studies with Src as a target for anti-cancer drugs, and numerous Src inhibitors have become available to test the importance of Src in tumor initiation and progression. In MM, it has been described that in cell lines and MM patient-derived tumors, c-Src is constitutively activated, which plays an important role in drug resistance mechanisms. Tris dibenzylideneacetone dipalladium (Tris DBA), a small-molecule palladium complex, was shown to reduce Src/NMT-1 complex in melanoma cells, as well as inhibit downstream signaling including mitogen-activated protein kinase (MAPK kinase) and phosphoinositol-3-kinase (PI3K). We suggest a novel strategy to improve the treatment of MM and overcome the drug resistance for the current therapeutic agents by specific inhibition of Src in MM cells by an organopalladium compound, Tris DBA. Methods: Tris DBA was prepared by Dr. Arbiser. MM cell lines (MM.1S, MM.1R, H929, RPMI-8826, and OPM2) and PBMCs were cultured with Tris DBA (0-10 µM) for 24h. MM cells were analyzed for cell proliferation by MTT assay; cell cycle by DNA staining with PI and analyzed by flow cytometry; apoptosis was analyzed by Annexin V/PI staining and analyzed by flow cytometry; and cell signaling associated with proliferation, cell cycle, and apoptosis was analyzed by western blotting. In addition, cell proliferation assay of Tris DBA with or without combination of proteasome inhibitors (PIs) bortezomib or carfilzomib for 24h was analyzed on the proliferation of MM cells in normoxic or hypoxic conditions. Moreover, we tested the effect of combination treatment on cell cycle and apoptosis signaling under normoxic conditions. We then evaluated the effect of Tris DBA on HIF1α expression, migration and drug resistance under normoxic or hypoxic conditions. Results: The Src inhibitor Tris DBA reduced the proliferation of MM cell lines with an IC50 of about 1.5 - 3 µM after 24h treatment as a single agent, while none of the normal PBMC controls showed effect on their proliferation in the same dose range. These results were consistent with the decreased expression of proliferation signaling proteins from MAPK pathways (pERK), as well as PI3K (pS6R). Src inhibition led to the induction of a sub-G1 peak, which indicated accumulating apoptotic cells shown by DNA staining with PI. Apoptosis was then analyzed by Annexin/PI and confirmed by cleavage of caspase-3 and PARP. We found that Tris DBA synergized with bortezomib and carfilzomib by inhibiting proliferation of MM cells and reducing cell cycle protein signaling more than either of the drugs alone. Moreover, the Tris DBA/Bortezomib or Tris DBA/Carfilzomib combination therapies significantly increased apoptosis by caspase-3 cleavage more than treatment with either proteasome inhibitor individually. Tris DBA inhibited HIF1α expression in both normoxic and hypoxic conditions. HIF1α is an important target for hypoxia-driven drug resistance. Our studies confirmed hypoxia promoted faster chemotaxis of MM cells towards the chemo-attractants found in stromal cell conditioned media, and that Tris DBA treatment could overcome this hypoxia-induced effect. In addition, the development of hypoxia-induced drug resistance to individual bortezomib or carfilzomib treatment was overcome with combination treatment of Tris DBA under hypoxic conditions. Conclusions: Tris DBA reduces proliferation and induces G1 arrest and apoptosis in MM cells. Tris DBA synergized with PIs reducing proliferation and cell cycle signaling, as well as increasing apoptosis more than each drug alone. Tris DBA overcame hypoxia-induced effects such as enhanced chemotaxis or drug resistance to PIs by inhibition of HIF1α expression. Moreover, we found that Tris DBA is an effective anti-myeloma agent alone or in combination with other targeted drugs and that it reverses hypoxia-induced drug resistance in myeloma. These results suggest the use of Tris DBA as a new therapeutic agent in relapsed refractory myeloma. Disclosures Arbiser: ABBY Therapeutics: Other: Jack L Arbiser is listed as inventor on a US Patent for imipramine blue. He is cofounder of ABBY Therapeutics, which has licensed imipramine blue from Emory University.. Azab:Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding.

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