Tumor Cell-Dependent Differences in Stroma-Induced Changes to Bortezomib Sensitivity.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2468-2468
Author(s):  
Eugen Dhimolea ◽  
Jana Jakubikova ◽  
Richard W.J. Groen ◽  
Jake E. Delmore ◽  
Hannah M. Jacobs ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Cell Response ◽  
Research Funding ◽  
Bioluminescence Imaging ◽  
Bortezomib Treatment ◽  
Rpmi 8226

Abstract Abstract 2468 In multiple myeloma (MM) and other hematologic malignancies, bone marrow stromal cells (BMSCs) confer resistance to diverse conventional or investigational therapeutics. During the last decade, data from many groups have concurred that the in vitro anti-MM activity of the proteasome inhibitor bortezomib is very similar in the presence and absence of BMSCs, including primary and immortalized BMSCs. These well-validated observations have supported the notion that novel, more effective, therapies for the treatment of MM should ideally be, similarly to bortezomib, capable of overcoming the protective effect of BMSCs. Interestingly, however, we have observed that primary CD138+ MM tumor cells isolated from patients with clinical refractoriness to bortezomib occasionally exhibit substantial in vitro response to clinically achievable concentrations of this drug. We therefore hypothesized that, under certain previously under-explored experimental settings, BMSCs may alter the threshold of MM cell response to bortezomib-induced apoptosis. To address this hypothesis in conditions that better simulate the clinical context, we conducted compartment-specific bioluminescence imaging (CS-BLI) assays to evaluate the effect of bortezomib on tumor cells co-cultured with BMSCs for different time periods prior to bortezomib administration. We observed that prolonged tumor-stromal co-culture (48–96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for several MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Prolonged co-culture of OPM1, RPMI-8226-Dox40, OCI-My5, KMS12BM and KMS18 cells prior to bortezomib treatment enhanced its activity. Importantly, extended co-culture of MM cell lines MM.1S and MM.1R with BMSCs prior to drug treatment induced significant attenuation of their response to bortezomib, as evidenced by 2–3 fold increase of IC50 values in several independent replicate experiments and a mean % area under the bortezomib dose response curve (AUC) of 5.82% vs 14.10% in the absence vs. presence of BMSCs, respectively (p=0.0079). Consistent with these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells mixed with Luc- BMSCs did not show statistically significant reduction in MM burden with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.1320), as quantified by bioluminescence imaging. In contrast, the same dose and schedule of bortezomib treatment significantly suppressed tumor burden, compared to vehicle-treated controls, of monotypic s.c. xenografts of Luc+ MM.1S cells in SCID mice (p=0.0022), as in prior experience. To evaluate the molecular mechanisms of cell non-autonomous decrease in MM cell response to bortezomib, we compared the transcriptional profiles of MM.1S cells in extended co-cultures with HS-5 BMSCs vs. MM.1S cells cultured in isolation. These studies identified a distinct transcriptional signature of stroma-induced transcripts, including several (e.g. PSMC3, ITGB7, FOS, ALDH1L2) for which transcript expression higher than the median levels for refractory MM patients correlated with shorter overall survival (p<0.02, log-rank tests) after treatment with bortezomib. These observations highlight the notion that tumor cell responses to a given agent in the presence of non-malignant stromal cells can exhibit substantial qualitative and quantitative variation, depending on the specific tumor cell type tested, as well as the particular stromal cell population and conditions of the co-culture. Our findings highlight the need to apply combinatorial high-throughput scalable platforms, such as CS-BLI, to evaluate the different permutations of interactions between tumor cells, non-malignant accessory cells of the microenvironment and administered therapeutics. This study also provides a comprehensive functional oncogenomic framework to identify prognostically relevant molecular mediators of stroma-induced resistance to therapy in MM. Disclosures: Groen: Genmab BV: Research Funding. McMilllin:Axios Biosciences: Equity Ownership. Mitsiades:Millennium Pharmaceuticals: Honoraria; Celgene: Honoraria; Novartis Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Merck &Co.: Honoraria; Centocor: Honoraria; Arno Therapeutics: Honoraria; Amgen: Research Funding; AVEO Pharma: Research Funding; OSI: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Johnson & Johnson: Research Funding; PharmaMar: Licensing royalties Other; Axios Biosciences: Uncompensated Role as advisor, Uncompensated Role as advisor Other.

Essential experimental steps and estimates of renal carcinoma initiating cells.

2014 ◽  
Vol 32 (15_suppl) ◽  
pp. 11127-11127
Author(s):  
Craig Gedye ◽  
Danylo Sirskyj ◽  
Nazleen Carol Lobo ◽  
Ella Hyatt ◽  
Andrew Evans ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Viability ◽  
Tumor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Rock Inhibitor ◽  
Cell Renal Cell Carcinoma ◽  
Cell Potential

11127 Background: Rare cancer stem cells (CSC), proposed to be solely responsible for tumor propagation and re-initiation, are functionally identified as tumor-initiating cells (TIC) from ex vivo tumors using xenotransplantation and clonogenic limiting dilution assays (LDA). TIC have not previously been described from ex vivohuman clear cell renal cell carcinoma (ccRCC). Methods: Primary human ccRCC samples (n=120) from patients undergoing nephrectomy were processed and implanted as subcapsular fragments or cell suspension injection LDAs with Matrigel in NOD/SCID/IL2Rγ-/- (NSG) mice, and observed for at least 6 months. In vitro clonogenic LDAs assays were performed from primary cell suspensions and ccRCC cell lines. LDAs were supplemented with human stromal cells and proteins, and the Y-26732 ROCK inhibitor. Multiparametric flow cytometry and immunofluorescence were used to investigate tumor heterogeneity and cell viability. Results: ccRCC TIC appeared rare from injected suspensions, but xenografts engrafted frequently from tiny fragments, and clonogenic frequencies were 103-104greater than TIC frequencies, suggesting that LDAs underestimated ccRCC tumor cell potential. We systematically identified multiple methodological steps that distort quantitation and identification of ccRCC TIC. For example cell viability was highly variable prior to processing, disaggregation itself destroyed up to 99% of tumor cells, standard assays substantially overestimated tumor cell viability in suspensions, and supplementation with human extracellular cells or proteins, or inhibition of anoikis by Y-26732 increased clonogenic and TIC frequencies in cell lines and primary ccRCC suspensions. Annexin-V staining revealed that tumor cells were more apoptotic then normal stromal cells, and that tumor cells positive for CD44 (a putative CSC marker) were more viable than CD44- tumor cells. Conclusions: We describe multiple, unappreciated and largely unavoidable observational errors in essential methods used to study TIC in ccRCC. ccRCC TIC may be more common than appreciated. Re-examination of the CSC hypothesis in other solid tumors is warranted in view of these previously unexplored methodological biases.

Cell Non-Autonomous Modulation Of Tumor Cell Responses To Pharmacological Inhibition Of The Proteasome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4454-4454
Author(s):  
Eugen Dhimolea ◽  
Richard W.J. Groen ◽  
Catriona A. Hayes ◽  
Jana Jakubikova ◽  
Bariteau Megan ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Tumor Cells ◽  
Cell Lines ◽  
Induced Resistance ◽  
Proteasome Inhibitor ◽  
Bioluminescence Imaging ◽  
Proteasome Inhibition ◽  
Bortezomib Treatment

Extensive preclinical studies of several groups using tumor cells co-cultured with bone marrow stromal cells (BMSCs) has documented that the potent anti-MM activity of the proteasome inhibitor bortezomib is not suppressed by BMSCs (e.g. primary and immortalized BMSCs). Using our compartment-specific bioluminescence imaging (CS-BLI) assays, we extended these observations to larger panels of MM cell lines. We observed, however, a recurrent pattern that primary CD138+ MM tumor cells from bortezomib-refractory patients recurrently exhibited substantial in vitro response to clinically-achievable concentrations and durations of bortezomib treatment. To simulate this clinicopathological observation, MM.1R-Luc+ cells were injected i.v. in SCID-beige mice and treated with bortezomib (0.5 mg/kg s.c. twice weekly for 5 weeks): diffuse MM tumors initially responded to bortezomib, but eventually became refractory. These in vivo-resistant MM cells were isolated from the mice and were treated in vitro with bortezomib, exhibiteing similar responsinveness to this agent as their isogenic bortezomib-naive MM cells, To further address the possibility that this represents a previously underexplored form of a microenvironment-induced alteration in bortezomib responsiveness, we studied how MM cells respond to pharmacological proteasome inhibition after variable times of co-culture with BMSCs prior to bortezomib exposure. We observed that prolonged tumor-stromal co-culture (48-96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for many of the MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Notably, prolonged co-cultures with BMSCs prior to bortezomib treatment enhanced the activity of this agent for other MM cell lines (e.g. OPM1, Dox40, OCI-My5, KMS12BM or KMS18). However, MM.1S and MM.1R cells, when exposed to extended co-cultures with BMSCs prior to initiation of drug exposure, exhibited significant attenuation (2-3 fold increase of IC50 values) of their response to bortezomib in several independent replicate experiments. In support of these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells co-implanted with Luc-negative BMSCs did not show significant reduction in MM tumor growth with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.13), as quantified by bioluminescence imaging. In co-cultures with BMSCs, MM.1S and MM.1R cells also exhibited suppression of their response to carfilzomib (the degree of this stroma-induced resistance was more pronounced that in the case of bortezomib for these 2 cell lines). Consistent with these observations, in vivo administration of carfilzomib in the orthotopic model of diffuse bone lesions of MM.1R-Luc+ cells was associated with less pronounced reduction in tumor growth, compared to bortezomib treatment (p<0.03). These results suggest that the stroma-induced attenuation of activity against a subset of MM cells represents a class-effect for this group of therapeutics, despite quantitative differences between different proteasome inhibitors. Mechanistically, we determined a distinct transcriptional signature of stroma-induced transcripts which are overexpressed in refractory myeloma patients with significantly shorter overall survival (p<0.03, log-rank tests) after bortezomib treatment. Our results in vitro and in vivo support the notion that the responses of MM cells to proteasome inhibition can exhibit substantial plasticity depending on the specific microenvironmental context with which these MM cells interact. We also identify prognostically-relevant candidate molecular mediators of stroma-induced resistance to proteasome-inhibitor based therapy in MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Breast Tumor Cell Lines From Pleural Effusions2

1974 ◽  
Vol 53 (3) ◽  
pp. 661-674 ◽  
Author(s):  
R. Cailleau ◽  
R. Young ◽  
M. Olivé ◽  
W. J. Reeves
Keyword(s):  
Chromosome Number ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Breast Tumor ◽  
Tumor Cell Lines ◽  
Breast Cancer Patients ◽  
Pleural Effusions ◽  
Epithelial Tumor

Summary During 1973, 4 new epithelial tumor cell lines were isolated from pleural effusions from breast cancer patients. We describe 3 of these lines: MDA-MB-134, with a mean chromosome number of 43; MDA-MB-175, with a mean chromosome number of 49; and MDA-MB-231, with a mean chromosome number between 65 and 69. We isolated the same cell type from 4 of 10 effusions from MDA-MB-134 and from 6 of 8 effusions from MDA-MB-175. We found that pleural effusions as a source of breast tumor cells to be cultured and studied in vitro have the following advantages: 1) large amounts of material and the possibility of obtaining sequential samples from the same patient; 2) high viability of tumor cells; 3) scarcity or absence of fibroblasts; and 4) the possibility of separating the tumor cells from other “contaminating” cell types by differences in their speed or degree of attachment to the flask. All lines from different patients differed, as seen grossly and microscopically. All lines from sequential pleural effusions from the same patient were apparently alike. No viruses or mycoplasmas were detected in any line.

Defibrotide (DF) Targets Tumor-Microenvironmental Interactions and Sensitizes Multiple Myeloma and Solid Tumor Cells to Cytotoxic Chemotherapeutics.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 286-286 ◽  
Author(s):  
Constantine S. Mitsiades ◽  
Cecile Rouleau ◽  
Krishna Menon ◽  
Beverly Teicher ◽  
Massimo Iacobelli ◽  
...  
Keyword(s):  
Stem Cell ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Solid Tumor ◽  
Single Agent ◽  
Conventional Chemotherapy ◽  
Adhesive Properties

Abstract Introduction: Defibrotide (DF) is a polydisperse oligonucleotide with anti-thrombotic, thrombolytic, anti-ischemic, and anti-adhesive properties, which selectively targets the microvasculature and has minimal hemorrhagic risk. DF is an effective treatment for veno-occlusive disease (VOD), an important regimen-related toxicity in stem cell transplantation characterized by endothelial cell injury. DF also augments stem cell mobilization by modulating adhesion in vivo. Because of its cytoprotective effect on the endothelium, we specifically investigated whether DF protects tumor cells from cytotoxic anti-tumor agents. Further, because of its broad anti-adhesive properties, we evaluated whether DF modulates the interaction of MM cells with bone marrow stromal cells (BMSCs), which confers growth, survival and drug resistance in the BM milieu. Methods: In vitro studies in isogenic dexamethasone (Dex)-sensitive and resistant MM cell lines (MM-1S and MM1R, respectively) showed that DF does not attenuate the sensitivity of MM cells to Dex, the proteasome inhibitor bortezomib (PS-341), melphalan (MEL), vinca alkaloids (vincristine, vinblastine), taxanes (paclitaxel) or platinum (cisplatin), but does decrease their sensitivity to doxorubicin. These selective effects in vitro of DF in protecting tumor cells against doxorubicin and modestly sensitizing MM cells to platinum was also confirmed in solid tumor breast (MCF-7) and colon (HT-29) carcinoma cell lines. Although DF had minimal in vitro inhibitory effect on MM or solid tumor cell growth in vitro, it showed in vivo activity as a single agent and enhanced the responsiveness of MM tumors to cytotoxic chemotherapeutics, such as MEL or cyclophosphamide, in human MM xenografts in SCID/NOD mice. The in vivo single-agent activity and chemosensitizing properties of DF, coupled with its lack of major in vitro activity, suggested that DF may not directly target tumor cells, but rather modulate tumor cell interaction with BMSCs. In an ex vivo model of co-culture of primary MM tumor cells with BMSCs (which protects MM cells against conventional chemotherapy), DF alone had a only modest effect on tumor cell viability, but it significantly enhanced MM cell sensitivity to cytotoxic chemotherapy (e.g. MEL), suggesting that a major component of the biological effects of DF may be attributable not to direct targeting of tumor cells, but to modulation of the interactions that tumor cells develop with the local stromal milieu. Conclusion: Our studies show that DF mediates in vivo anti-MM activity by abrogating interactions of MM cells with their BM milieu, thereby enhancing sensitivity and overcoming resistance to conventional chemotherapy. These data support future clinical trials of DF, in combination with both conventional and novel therapies, to improve patient outcome in MM.

Novel Targeting Of Phospho-Marcks Overcomes Drug Resistance and Induces Anti-Tumor Activity In Preclinical Models Of Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 282-282 ◽  
Author(s):  
Yijun Yang ◽  
Manujendra N Saha ◽  
Yan Chen ◽  
Lugui Qiu ◽  
Donna E Reece ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Resistance ◽  
Cell Lines ◽  
Research Funding ◽  
Resistant Cell ◽  
Drug Resistant ◽  
Sensitive Cell ◽  
Bortezomib Treatment ◽  
Cell Cycle Pathway ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) remains incurable due to the development of a drug-resistant phenotype after prolonged therapy. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a protein kinase C (PKC) substrate that plays an important role in cell adhesion, spreading and invasion. Our previous studies found that overexpression of phospho-MARCKS (pMARCKS) was detected in developed drug resistant MM cell lines (RPMI-8226 R5, MM.1R) relative to their parental drug sensitive cell lines (RPMI-8226S, MM.1S). We hypothesized that pMARCKS is involved in chemo- and novel drug resistance in MM. To further evaluate the drug resistance, we exposed both RPMI-8226 R5 and MM.1R cell lines to varying dosages of bortezomib, dexamethasone, doxorubicin, and lenalidomide. By MTT assay, both resistant cell lines were found to have significantly higher viability to all 4 drugs compared to their respective non-resistant lines. In addition, Western blot analysis showed increased pMARCKS expressions in all 3 bortezomib resistant cell lines 8226.BR, OPM2.BR, and ANBL-6.BR as compared to their respective bortezomib sensitive cell lines. We next acquired MM patient samples collected at diagnosis and at relapse after bortezomib treatment, and investigated their pMARCKS expression with immunoblotting analyses. The patient samples collected from relapse after bortezomib treatment had higher pMARCKS expression than those collected at diagnosis. Furthermore, we studied additional 3 primary MM patient samples with high pMARCKS expressions and 3 with low expressions for their vaibility after a 36 hour bortezomib treatment, and found that the samples with high pMARCKS expressions were more resistant to bortezomib than those with low pMARCKS expressions (mean IC50 of 7.1 nM and mean IC50 of 4.8 nM, respectively; p = 0.042). Importantly, combination of a low dosage of bortezomib (5.0 nM) with either 2.5 uM or 5.0 uM of enzastaurin (an inhibitor of phospho-PKC), displayed a synergistic cytotoxicity on myeloma cells with high pMARCKS expressions. To further elucidate the role of pMARCKS in drug resistance, we knocked down pMARCKS expression by transfecting siMARCKS into 8226 R5 and MM.1R cells. Following the knockdown, both cell lines had significantly lower viability after treatment with either bortezomib, dexamethasone, doxorubicin, or lenolidomide, in comparison to empty vector controls. FACS analysis and annexin V assay of the knockdown cells and the control cells from both cell lines showed significantly induction of G1/S cell cycle arrest and apoptosis in the knockdown cells. The immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) DNA-qPCR analysis further demonstrated that pMARCKS regulates SKP2 expression through binding with E2F1, mediating SKP2/p27Kip1 cell cycle pathway. Finally, we investigated the effect of inhibition of pMARCKS in a 8226 R5 xenograft model of SCID mice. Mice injected with shMARCKS-transfected 8226 R5 cells and received bortezomib showed significant retardation of tumor growth and prolonged survival compared to the control groups. Taken together, our data indicate that pMARCKS is constitutively activated in resistant and relapsed MM cells and contributes to drug resistance by regulating E2F1 mediated SKP2/p27Kip1 cell cycle pathway, thus providing a preclinical rationale for targeting pMARCKS as a promising approach in patients with refractory/relapsed MM. Disclosures: Reece: BMS: Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Millennium: Research Funding; Novartis: Honoraria; Onyx: Honoraria.

Silencing The Sialyltransferase Gene ST3GAL6 Inhibits Adhesion and Migration Of Myeloma Cells In Vitro and Reduces The Homing and Proliferation Of Tumor Cells In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 275-275
Author(s):  
Siobhan Glavey ◽  
Salomon Manier ◽  
Antonio Sacco ◽  
Michaela R Reagan ◽  
Yuji Mishima ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Board ◽  
Myeloma Cells ◽  
Sialyl Lewis ◽  
And Migration ◽  
Rpmi 8226

Abstract Background Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The adhesion and trafficking of multiple myeloma (MM) cells is strongly influenced by glycosylation and multiple myeloma cells express a variety of adhesion molecules, including selectin ligands and integrins, which are typically dependent on glycosylation for their function. We have previously reported that the sialyltransferase ST3GAL6 is up-regulated in plasma cells from MM patients and that increased expression is associated with inferior overall survival (OS) in MM gene expression profiling (GEP) datasets. The functional significance of increased sialylation of MM cells has not previously been reported. Methods MM cell lines MM1s and RPMI-8226 were confirmed to have high expression levels of ST3GAL6 at the gene and protein level compared to healthy controls. Knockdown of ST3GAL6 was confirmed in MM cell lines RPMI-8226 and MM1s using lentiviral shRNAs targeting different regions in the ST3GAL6 mRNA. Specific ST3GAL6 knockdown was confirmed by reduced ST3GAL6 mRNA and protein expression in comparison to a scrambled control. In a calcein-AM fluorescence based adhesion assay we next evaluated the effects of ST3GAL6 knockdown on MM-cell adhesion to bone marrow stromal cells (BMSC’s) and fibronectin coated plates. Migration to 30nM SDF1-α was assessed using transwell plates comparing ST3GAL6 knockdown cells to scrambled controls. The commercially available sialyltransferase inhibitor 3Fax-Neu5Ac was used to pre-treat MM cells in vitro prior to assessment of apoptosis by flow cytometry. shST3GAL6 MM1s cells positive for green fluorescent protein and luciferin (GFP-Luc+) were injected into tail veins of SCID-Bg mice (5x106 cells, n=5/group) and mice were followed weekly using bioluminescent imaging (BLI) for tumor development. Bone marrow homing of tumor cells was assessed using in vivoconfocal imaging of the skull vasculature (n=3/group). Results Knockdown of ST3GAL6 in MM cell lines resulted in a 50% reduction in cell surface staining with the monoclonal antibody HECA-452. This indicated reduced expression of cutaneous lymphocyte associated antigen (CLA), a carbohydrate domain shared by sialyl Lewis X (sLex) and sialyl Lewis a (sLea) antigens, confirming suppression of ST3GAL6 activity. There was a significant reduction in the ability of knockdown cells to adhere to BMSC’s and fibronectin in-vitro compared to scrambled controls (P=0.016, 0.032 respectively). Migration ability of these cells in response to SDF1-α was also reduced (P=0.01). In vivo in a xenograft SCID-Bg mouse model shST3GAL6 cells demonstrated a reduced tumor burden as assessed by weekly BLI (P=0.017 at week 4). A consolidated map of the skull bone marrow niche in mice injected with shST3GAL6 MM1s GFP-Luc+ cells revealed a reduced homing ability of these cells in comparison to mice injected with scrambled control cells. Treatment of the MM cell lines MM1s and RPMI-8226 with a sialyltransferase inhibitor 3Fax-Neu5Ac resulted in almost complete elimination of cell surface sLex and/or sLea expression as determined by HECA-452 staining. Following pre-treatment with 3Fax-Neu5Ac, MM1S cells grown in co-culture with BMSC’s cells showed increased sensitivity to Bortezomib compared to cells treated with bortezomib alone. Conclusions shRNA knockdown of ST3GAL6 in MM cells significantly inhibits adhesion and migration in vitro with reduced homing and proliferation potential in vivo. In conjunction with the results of enzymatic inhibition this indicates that sialylation may play an important role in the malignant behavior of MM cells. Studies are ongoing to address the potential role of altered glycosylation in MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Evaluation Of The Novel, Orally Bioavailable Selective Inhibitor Of Nuclear Export (SINE) Verdinexor (KPT-335) In Spontaneous Canine Cancer: Results Of Phase I and Phase II Clinical Trials

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5149-5149
Author(s):  
Cheryl London ◽  
Luis feo Bernabe ◽  
Sandra Barnard ◽  
William Kisseberth ◽  
Antonella Borgatti ◽  
...  
Keyword(s):  
Biological Activity ◽  
Tumor Cell ◽  
Median Time ◽  
Cell Lines ◽  
Nuclear Export ◽  
Research Funding ◽  
Tumor Cell Lines ◽  
Time To Progression ◽  
Orally Bioavailable

Abstract SINE (Selective Inhibitors of Nuclear Export) block the activity of XPO1/CRM1, 1 of 7 nuclear export proteins in cells, forcing the nuclear retention of key tumor suppressor proteins (TSP), leading to selective apoptosis of tumor cells.  The purpose of these studies was to evaluate the in vitro activity of SINE against canine tumor cell lines and investigate the biological activity of Verdinexor (KPT-335) in dogs with spontaneous cancers as proof of principle for human clinical studies with SINE. Several different canine tumor cell lines including those derived from Non-Hodgkin Lymphoma (NHL) exhibited growth inhibition and apoptosis in response to nanomolar concentrations of SINE; NHL cells were particularly sensitive with IC50 concentrations ranging from 2-42 nM. A Phase I clinical trial of Verdinexor was performed in dogs with cancer with an emphasis on NHL given in vitro activity demonstrated against the tumor cell lines. The maximum tolerated dose (MTD) was 1.75 mg/kg twice per week although biological activity was observed at 1 mg/kg. Clinical benefit including Partial Response (PR) and Stable Disease (SD) for at least 4 weeks was observed in 9/14 dogs with NHL with a median time to progression of 66 days (range 35-256). A dose expansion study was performed in 6 dogs with NHL given 1.5 mg/kg Verdinexor on a Monday/Wednesday/Friday (MWF) regimen; clinical benefit (PR + SD) was observed in 4/6 dogs with a median time to progression of 83 days (range 35-250+). Toxicities were primarily gastrointestinal in nature consisting of anorexia, weight loss, vomiting and diarrhea and were manageable with supportive care and dose modulation.  A validated health related Quality of Life (QOL) form used to assess dogs during treatment demonstrated that the overall quality of life did not decrease in dogs in this study supporting the notion that clinical toxicities associated with Verdinexor are generally well tolerated.  Based on these findings, a Phase IIb study was performed in 58 dogs with either newly diagnosed or relapsed NHL.  Drug was administered initially at 1.5 mg/kg MWF, but this dosing regimen was changed to 1.25 mg/kg M/Th due to the high rate of anorexia and weight loss on the MWF regimen; dose escalation was permitted to 1.5 mg/kg on the M/Th regimen.  The objective response rate was 34% (1 CR, 19 PR) with an additional 33 dogs experiencing SD for a minimum of 4 weeks, resulting in a of 91% disease control rate. While the median time to progression was approximately 5 weeks, 19 dogs (32%) remained on study drug for >8 weeks; several dogs continue to receive Verdinexor.  Laboratory abnormalities were minimal and clinical toxicities were mild on the M/Th regimen.  Together, these data provide robust evidence that the novel orally bioavailable XPO1 inhibitor Verdinexor exhibits single agent biological activity in a spontaneous large animal model of human NHL. Furthermore, Verdinexor was well tolerated even in the absence of supportive care, suggesting that SINE compounds could exhibit good long-term tolerability in people. Disclosures: London: Zoetis: Honoraria, Research Funding; Karyopharm: Consultancy, Research Funding; Abbott: Honoraria. Modiano:Karyopharm: Research Funding. Saint-Martin:Karyopharm: Employment. McCauley:Karyopharm : Employment, Equity Ownership, Patents & Royalties. Shacham:Karyopharm : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment.

scholarly journals PIM Kinase Inhibition Decreases the Proangiogenic Properties of Multiple Myeloma Cells and Affects the Metabolic State of the Vascular Endothelium

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 16-17
Author(s):  
Filip Garbicz ◽  
Anna Szumera-Ciećkiewicz ◽  
Joanna Barankiewicz ◽  
Dorota Komar ◽  
Michał Pawlak ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Cell Death ◽  
Tumor Cell ◽  
Cell Lines ◽  
Fold Increase ◽  
Pim Kinases ◽  
Rpmi 8226

The development and progression of multiple myeloma (MM) depend on the formation and perpetual evolution of an immunosuppressive and hypervascular bone marrow microenvironment. MM undergoes an angiogenic switch during its early progression stages and initiates the secretion of proangiogenic proteins, such as VEGFA and Galectin-1. Following their engagement with the VEGF receptor 2 on the surface of the endothelium, quiescent endothelial cells (ECs) rapidly switch to an activated state, thus gaining the ability to create sprouts, migrate and proliferate. However, chronic angiogenic stimulation results in the formation of a dense and leaky network of pathological vessels, which in the case of MM also serves as a major source of prosurvival paracrine signals. Since PIM kinases are known modulators of cytokine signaling, owing to their ability to activate NFκB, JAK/STAT and mTOR pathways, we analyzed the expression pattern of PIM1, PIM2 and PIM3 in multiple myeloma bone marrow samples using immunohistochemistry. We found that both MM cells as well as myeloma-associated ECs exhibit a significantly higher PIM3 expression than their normal bone marrow counterparts. Since the role of PIM kinases in the vascular compartment of the tumor microenvironment is currently unknown, we decided to explore the proangiogenic functions of PIM kinases using in vitro MM and EC model cell lines. 3 MM cell lines (RPMI 8226, MM1.s, U266), immortalized bone marrow ECs (HBMEC-60) and human umbilical vein ECs (HUVECs) were used for the experiments. Primary MM cells were obtained from MACS-separated bone marrow aspirates. Chemical blockade of PIM kinase activity was achieved using the pan-PIM inhibitor SEL24/MEN1703. The compound decreased the viability of MM cell lines with IC50 in the submicromolar range, induced G2 cell cycle arrest and apoptosis. Moreover, SEL24/MEN1703 induced apoptosis in primary MM cells, even when cocultured with the CD138- bone marrow fraction. PIM inhibitor treatment inhibited the phosphorylation of mTOR substrates S6 and 4EBP1, STAT3/5, as well as RelA/p65. Consequently, we observed markedly decreased VEGFA and Gal-1 levels in SEL24/MEN1703-treated MM cells. When cultured together, separated by a permeable transwell membrane, both RPMI 8226 cells, as well as ECs, exhibited a 2-fold increase in proliferation rate. This effect was completely blocked by a 2-day treatment with a PIM inhibitor. Exposure of ECs to recombinant VEGFA (10ng/ul) or MM supernatant resulted in an increase in VEGFR2 Y1175 phosphorylation level and induction of PIM3 expression. Increased MYC activity is a hallmark of VEGF-dependent endothelial activation and is necessary to support the creation of new vessels. Since the PIM3 promoter region contains putative MYC-binding sites (E-boxes), we checked if PIM3 induction depends on MYC in ECs. MYC silencing using siRNA resulted in an 88% lower PIM3 expression than the non-targeting siRNA. One of MYC's main tasks during angiogenesis is the stimulation of cellular ATP synthesis to meet the energy demands created by the dynamic remodeling of the actin cytoskeleton. Surprisingly, PIM inhibition decreased the total ATP content in ECs by 25%, thus disrupting the energetic homeostasis, as evidenced by a 9.6-fold increase in phosphorylated AMPK T172 levels. Furthermore, SEL24/MEN1703-treated ECs were depleted of higher-order actin structures necessary for efficient angiogenesis, such as actin stress fibers, membrane ruffles and lamellipodia. In consequence, PIM kinase inhibition decreased proliferation, migration and formation of new vessel-like structures in Matrigel by ECs. Collectively, our data demonstrate that PIM inhibition induces MM cell death and abolishes important tumor cell-ECs interactions. In addition, we show that PIM3 is overexpressed in MM tumor endothelial cells and PIM inhibition disrupts the activation state in in vitro cultured ECs. Hence, targeting PIM kinases may represent an efficient approach to induce tumor cell death and to block angiogenesis in MM. RNA-sequencing studies on the downstream effectors of PIM3 are currently ongoing in order to unravel the molecular mechanism behind the observed effects. Figure Disclosures Brzózka: Ryvu Therapeutics: Current Employment. Rzymski:Ryvu Therapeutics: Current Employment. Tomirotti:Menarini Ricerche: Current Employment. Lech-Marańda:Roche, Novartis, Takeda, Janssen-Cilag, Amgen, Gilead, AbbVie, Sanofi: Consultancy; Roche, Amgen, Gilead: Speakers Bureau. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.

scholarly journals STUDY OF EFFECTS OF CIRCADIAN RHYTHMS ON SOLID TUMOR CELLS PROLIFERATION AND CHEMORESISTANCE

2020 ◽  
Vol 66 (5) ◽  
pp. 563-571
Author(s):  
Anna Danilova ◽  
N. Avdonkina ◽  
Ye. Gubareva ◽  
I. Baldueva ◽  
Anton Zozulya ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Solid Tumor ◽  
Lung Tumor ◽  
Morphological Characteristics ◽  
Biological Properties ◽  
Physiological Processes

Circadian clock is a complex mechanism regulating many different physiological processes. Preclinical, epidemiological and clinical studies demonstrate association between circadian rhythms disruption and tumor initiation. Study of modulation of solid tumor cells biological properties through enhancement of clock mechanisms could attribute to the development of more effective chemo- and hormone therapy approaches. Aim: Evaluate the effects of ovarian and lung tumor cells synchronization with dexamethasone in vitro on cells sensitivity to cisplatin. Materials and methods: Metastatic ovarian cancer (n=3) and lung cancer (n=3) cell lines were obtained from patients tumors. Tumor cell cultivation was performed in accordance with the protocol. Artificial synchronization was performed with dexamethasone 200 nM introduction to the cell cultures. Doses of cisplatin used were 1.5 and 3.0 mg/ml. xCELLigence Real-Time Cell Analysis and Cell-IQ was used to measure proliferation and chemoresistance of tumor cells. Results: Each cell-line had individual morphological characteristics and proliferation parameters. Preliminary incubation with dexamethasone (2 h) had a stimulating effect on proliferation of all tumor cell lines (Slope min -4.3(0.3)хЕ ‘х10-3 - max 36.8(0.6)хЫх10'3, min 2.2(0.2)хЕ1х10'3- max 50.4(0.8)хЕ1х10'3), and increased their sensitivity to cisplatin (min -43(2.6)хЕ1х10-3 - max 57.5(0.6)хЕ1х10-3 и min -217,3(2,2) -1,9(0,1)хч-1х10-3 - max -1,9(0,1)хч'1х10'3, respectively. Conclusion: These results should be the platform for future studies of the interaction of clock mechanisms, cell cycle regulation and viability of tumor cells.

Virus like Particles in a Human Lung Tumor

1974 ◽  
Vol 32 ◽  
pp. 18-19
Author(s):  
R.E. Nordquist ◽  
R.E. Coalson ◽  
J.A. Mohr ◽  
E.R. Rhoades ◽  
J.J. Coalson ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Lung Tumor ◽  
Biological Agent ◽  
Virus Like Particles ◽  
Human Lung Tumor ◽  
Ultrastructural Studies ◽  
Indicator Cell

Ultrastructural studies of pulmonary needle biopsies from patients with alveolar cell carcinoma (ACC) revealed the presence of a virus-like material in the nucleus and cytoplasm of tumor cells. Stinson et. al. reported the presence of filamentous virus-like particles in four of six cases in an ultrastructural study of ACC. In vitro studies by Coalson et. al. confirmed the presence of a biological agent and demonstrated that cell-free supernate from tumor cell cultures could induce cytopathic effect when applied to indicator cell lines. It was also shown in this report that tumor cell lines derived from this tumor produced the filterable biological agent until the 18-20 passage in culture. An extension of this investigation on the cell lines derived from ACC demonstrated that a unique antigen was associated with ACC tumor cells and that this antigenicity could be induced in indicator cell lines following treatment with cell free extracts.

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