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.

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.

The Novel Potent Pan-Deacetylase (DAC) Inhibitor, Panobinostat (LBH589), Markedly Enhances the Anti-Myeloma Effects of Chemotherapy In Vitro and In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2520-2520
Author(s):  
Richard A. Campbell ◽  
Eric Sanchez ◽  
Jeffrey Steinberg ◽  
Michael Share ◽  
Joseph Wang ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Low Dose ◽  
Liposomal Doxorubicin ◽  
Single Agent ◽  
Mouse Serum ◽  
Once Daily ◽  
Treatment Groups

Abstract Deacetylase (DAC) inhibitors represent a new class of anti-cancer therapeutics that inhibit DAC enzymes and have been shown to have multiple effects in tumor cell lines including decreased oncoprotein expression (Bcr-Abl, HER-2), decreased angiogenesis, induction of apoptosis, induction of cell-cycle arrest, and decreased tumor cell motility and invasion. Panobinostat (LBH589), a novel cinnamic hydroxamic acid analogue with potent histone DAC inhibitor activity, has recently been shown to have the potential to treat a wide range of solid and hematological malignancies including multiple myeloma (MM). In this study, we first evaluated the in vitro anti-MM effects of panobinostat alone and in combination with doxorubicin or melphalan using the MM cell lines RPMI8226, U266 and MM1S. Cells treated with the combinations of panobinostat + doxorubicin and panobinostat + melphalan showed marked synergistic anti-MM effects as determined by measuring proliferation with the MTS assay compared to treatment with single agent and untreated cells. Next, we evaluated the anti-MM effects of panobinostat alone and in these combinations in vivo using one of our SCID-hu mouse models of human MM, LAGλ-1. Each SCID mouse was implanted with a 2.0 - 4.0 mm3 LAGλ-1 into the left superficial gluteal muscle. In our panobinostat single agent study, tumors were allowed to grow for 7 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into panobinostat treatment groups. Panobinostat was administered via intraperitoneal (i.p.) injection once daily five times per week at 5, 10 and 20 mg/kg. Control mice were given sterile normal saline as vehicle. Mice receiving panobinostat showed marked inhibition of tumor growth (10 mg/kg, P < 0.003; 20 mg/kg, P < 0.009) and reduction of paraprotein levels (10 mg/kg, P < 0.0025; 20 mg/kg, P < 0.015) compared to mice receiving vehicle. Next, we evaluated the combination of low-dose panobinostat (5 mg/kg) with low doses of either liposomal doxorubicin (1 mg/kg) or melphalan (3 mg/kg) i.p. in mice bearing LAGλ-1. Tumors were allowed to grow for 10 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into treatment groups. Panobinostat was administered as above, and liposomal doxorubicin was injected once daily for three consecutive days weekly and melphalan once weekly. Mice treated with the combination of panobinostat + liposomal doxorubicin showed markedly smaller tumors and reduced hIgG levels compared to treatment with the DAC inhibitor alone, and treatment with liposomal doxorubicin as a single agent produced no anti-MM effects. Mice bearing LAGλ-1 treated with the combination of low-dose panobinostat + low-dose melphalan also showed markedly smaller tumors and decreased hIgG levels compared to treatment with panobinostat alone whereas mice receiving melphalan alone showed similar results to vehicle-treated animals. These promising results support the further clinical development of panobinostat and suggest that combining this DAC inhibitor with low-dose chemotherapy (liposomal doxorubicin or melphalan) may enhance the efficacy of this novel agent for the treatment of MM patients.

Ofatumumab (OFA) Is a Novel Anti-CD20 Monoclonal Antibody (mAb) with Improved Anti-Tumor Activity in Vitro and in Vivo in Mantle Cell Lymphoma (MCL) Pre-Clinical Models.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2757-2757 ◽  
Author(s):  
Matthew J. Barth ◽  
Gopichand Pendurti ◽  
Ping-Chiao Tsai ◽  
Cory Mavis ◽  
Pavel Klener ◽  
...  
Keyword(s):  
Stem Cell ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Parp Cleavage ◽  
Clinical Models ◽  
Anti Cd20

Abstract Abstract 2757 MCL is typically characterized by an aggressive clinical course and inevitable development of refractory disease despite early intervention that often includes: immunotherapy (e.g., rituximab), multi-agent induction chemotherapy and consolidation with high dose chemotherapy and autologous stem cell transplant in first remission. Residual disease at the time of stem cell collection is an important cause for treatment failure. There is a need to evaluate more potent anti-CD20 mAbs capable to kill lymphoma cells with low CD20 surface levels. In Burkitt's lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) pre-clinical models we previously demonstrated that OFA was more potent than rituximab (RIT) in vitro and in vivo. In order to characterize the activity of OFA against MCL, we evaluated the activity of OFA against cytarabine (Ara-C)-sensitive (eg. Mino, Jeko-1, Rec-1, HBL-2, Granta and Z-138); –resistant MCL cell lines (eg. MinoAraCR, Jeko-1AraCR, Rec-1AraCR, HBL-2AraCR and GrantaAraCR); and primary tumor cells derived from MCL patients (n=2). Antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) were measured by standard 51Cr release assays in MCL exposed to OFA, RIT or isotype control. OFA vs. RIT direct anti-proliferative effects were measured in by alamar blue reduction assay. Apoptosis following in vitro exposure to OFA or RIT was detected by caspase 3/PARP cleavage. Patient tumor cells were isolated from biopsy specimens by negative selection using magnetic beads and incubated with OFA or RIT +/− human serum as a complement source. Cell viability was determined at 48 hours by CellTiterGlo assay. Surface CD20 and the complement inhibitory proteins (CIPs) (CD55 and CD59) density in MCL cell lines was determined by flow cytometry (Image stream) and compared to BL or DLBCL cell lines. For in vivo experiments 6–8 week-old SID mice were inoculated subcutaneously with 5×106 matrigel suspended Z-138 cells. Upon tumor engraftment, mice were assigned to RIT (10mg/kg), OFA (10mg/kg) or control groups. Tumor growth curves were calculated for each group. Mice were sacrificed if tumor size reached >2cm in any dimension. After 6 months, survival was analyzed by Kaplan-Meier analysis and compared by log-rank test. OFA induced significantly higher levels of CDC associated cell lysis compared to RIT in almost all MCL cell lines tested (10/11) (Mino: 53.2% vs 0.2%; MinoAraCR: 72.6% vs. 0.6%; Jeko-1: 33.4% vs. 9.8%; Jeko-1AraCR: 38.3% vs. 2.8%; REC-1: 17% vs 3%; Rec-1AraCR: 7.8% vs. 0.2%; HBL-2: 27.1% vs. 19.2%; HBL-2AraCR: 86.6% vs. 72.2%; GrantaAraCR: 17% vs 0.9%; Z-138: 56.4% vs. 0.65%; all p-values <0.05). No differences in RIT or OFA mediated ADCC or direct signaling was observed. As previously noted in BL and DLBCL models, OFA was capable of inducing a higher degree of CMC even at low CD20 levels in contrast to RIT. In vivo, OFA slowed tumor growth, and prolonged survival in Z-138 bearing SCID mice compared to RIT (median survival for RIT was 127 days vs. not reached for OFA treated animals; p<0.05). Our data suggest that, OFA is more potent than RIT against Ara-C-sensitive and –resistant MCL cells in vitro, delays tumor growth and prolongs survival compared to RIT in an in vivo MCL SCID mouse model, and retains CDC activity despite low CD20 and high CIP surface expression levels. OFA appears to be a promising mAb targeting CD20 in MCL and is undergoing clinical testing in the front-line setting (NCT01527149). Disclosures: Czuczman: Genmab: Unrestricted Research Grant Other.

GRP-78 secreted by tumor cells blocks the antiangiogenic activity of bortezomib

Blood ◽  
2009 ◽  
Vol 114 (18) ◽  
pp. 3960-3967 ◽  
Author(s):  
Johann Kern ◽  
Gerold Untergasser ◽  
Christoph Zenzmaier ◽  
Bettina Sarg ◽  
Guenther Gastl ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Solid Tumor ◽  
Chorioallantoic Membrane ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Tumor Xenografts

Abstract Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomib's inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts, thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy, we identified GRP-78, a chaperone protein of the unfolded protein response, as being responsible for bortezomib resistance. Indeed, a variety of bortezomib-resistant solid tumor cell lines (PC-3, HRT-18), but not myeloma cell lines (U266, OPM-2), were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal–related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data, we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment, thus demonstrating a hitherto unknown mechanism of resistance to bortezomib.

scholarly journals Human Placental CD34 +-Derived Natural Killer Cells with High Affinity and Cleavage Resistant CD16 (CYNK-101) in Combination with Daratumumab for Immunotherapy Against CD38 Expressing Hematological Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2773-2773
Author(s):  
Irene Raitman ◽  
Joseph Gleason ◽  
Salvatore Rotondo ◽  
Shuyang He ◽  
Valentina Rousseva ◽  
...  
Keyword(s):  
B Cells ◽  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Xenograft Model ◽  
Tumor Cell Lines ◽  
Publicly Traded

Abstract Natural Killer (NK) cells are key mediators of antibody dependent cellular cytotoxicity (ADCC) via the CD16 Fc receptor. NK cellular therapies can effectively be targeted to tumor antigens when combined with tumor specific antibodies. Celularity Inc. is developing human placental CD34 +-derived, cryopreserved, off-the-shelf, allogenic NK cells (CYNK-101) with a high IgG binding affinity and proteinase cleavage resistant CD16 variant (CD16VP) for cancer treatment. We hypothesize that expressing CD16VP augments anti-tumor ADCC activity. Reported here are the in vitro and in vivo results of evaluating CYNK-101 cytotoxicity against CD38 expressing multiple myeloma (MM) and lymphoma tumor cell lines when in combination with daratumumab, an anti-CD38 monoclonal antibody. Human placental CD34 + cells were transduced with lentivirus expressing CD16VP and cultured in the presence of cytokines to generate CYNK-101 cells. The in vitro cytotoxic activity of CYNK-101 against CD38 + MM (MOLP-8, LP-1, MM.1S) and lymphoma (Daudi) tumor cell lines, and normal B-cells was assessed in combination with daratumumab via flow cytometry based ADCC assays and cytokine secretion was assessed via multiplex Luminex analysis. In vivo ADCC activity of CYNK-101 was assessed using a disseminated B-cell lymphoma xenograft model in B-NDG-hIL15 mice. B-NDG-hIL15 mice lack T, B, and NK cells and are transgenic for human IL-15 to support CYNK-101 persistence and maturation. Luciferase expressing Daudi cells (3×10 6) were intravenously (IV) injected on Day 0 three days after preconditioning with a myeloablative dose of busulfan to allow for better tumor cell engraftment. CYNK-101 cells (1x10 7) and/or daratumumab (0.05 mg/kg) were IV injected on Days 7, 14 and 21. Tumor burden was assessed weekly by bioluminescence imaging (BLI) and the mice were followed for assessment of their survival (n=5 mice per group). In vitro ADCC studies indicate enhanced cytolysis of CYNK-101 in combination with daratumumab against both MM and lymphoma tumor cells compared to that of IgG control. At 24h at the effector to target (E:T) ratio of 5:1, CYNK-101 (n=5 donors) demonstrated a cytolysis of 87.6 ± 6.3% with daratumumab vs. 37.3 ± 9.5% with IgG control against MOLP-8 (p&lt;0.001), 73.9 ± 2.5% vs. 32.1 ± 7.2% against LP-1 (p&lt;0.001), 77.2 ± 11.5% vs. 67.4 ± 10.7% against MM.1S (p&lt;0.001), and 54.7 ± 24.0% vs. 4.3 ± 2.6% against Daudi (p&lt;0.01) tumor cells. Secretion of GM-CSF, IFN-γ, and TNF-α was increased in CYNK-101 co-cultured with the tumor cell lines in the presence of daratumumab for 24h (n=5 donors, p&lt;0.05). When cocultured with mixed LP-1 and CD38 + normal B-cells, CYNK-101 in combination with daratumumab displayed specific cytotoxicity against LP-1, while sparing CD38 + normal B-cells even at an E:T ratio up to 100:1, demonstrating that CYNK-101 can distinguish CD38 + tumor cells from CD38 + normal cells. Additionally, despite expression of CD38 on CYNK-101 there was no NK fratricide observed when CYNK-101 were in combination with daratumumab. In vivo studies in the lymphoma xenograft model revealed a significant decrease in tumor burden as evidenced from bioluminescence imaging at day 28 (1 week after last CYNK-101 injection) for mice that received CYNK-101 in combination with daratumumab compared to vehicle control (p&lt;0.001), CYNK-101 (p&lt;0.05) and daratumumab (p&lt;0.05). Furthermore, CYNK-101 in combination with daratumumab demonstrated an enhanced survival benefit with a median survival of 35 days versus a median survival of 28 days for the vehicle treated group (p&lt;0.005). In summary, our results demonstrate enhanced in vitro and in vivo ADCC activities of CYNK-101 in combination with daratumumab against CD38 + hematological tumors and warrant further development of this combination therapy for these cancers. Disclosures Raitman: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Gleason: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Rotondo: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. He: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Rousseva: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Guo: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Rana: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. van der Touw: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Ye: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Kang: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Hariri: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang: Celularity Inc.: Current equity holder in publicly-traded company, Ended employment in the past 24 months.

GRP-78 Secreted by Tumor Cells Blocks the Anti-Angiogenic Activity of Bortezomib.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4805-4805
Author(s):  
Johann Kern ◽  
Gerold Untergasser ◽  
Christoph Zenzmaier ◽  
Bettina Sarg ◽  
Gunther Gastl ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Solid Tumor ◽  
Conflicts Of Interest ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Tumor Xenografts

Abstract Abstract 4805 Anti-angiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane (CAM). Bortezomib's inhibitory effects on CAM vascularization were abrogated in the presence of distinct tumor xenografts thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy we identified GRP-78, a chaperone protein of the unfolded protein response, as being responsible for bortezomib resistance. In fact, a variety of bortezomib-resistant solid tumor cell lines (PC-3, HRT-18), but not myeloma cell lines (U266, OPM-2), were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib, but induced prosurvival signals by phosphorylation of ERK and inhibited p53-mediated expression of pro-apoptotic Bok and Noxa proteins in endothelial cells. From our data we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment, thus demonstrating a hitherto unknown mechanism of resistance to bortezomib. Disclosures: No relevant conflicts of interest to declare.

Preclinical Studies of Salinomycin In Multiple Myeloma (MM) Models: Targeting of Side Population (SP) Cells In the Context of Tumor – Microenvironment Interactions.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1574-1574
Author(s):  
Efstathios Kastritis ◽  
Jana Jakubikova ◽  
Jake Delmore ◽  
Steffen Klippel ◽  
Douglas W. McMillin ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cell ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Committees ◽  
Ic50 Values

Abstract Abstract 1574 Cancer cells with stem cell-like features are a topic of intense research because their resistance to existing drugs is considered a culprit for relapses, even in patients with complete remission defined by clinical, biochemical and imaging parameters or by sensitive molecular techniques. Salinomycin, an antibacterial and coccidiodostatic ionophore, is reported (Cell 2009;138(4):645-59) to be >100-fold more potent against breast cancer cells with stem cell-like phenotype after mesenchymal transdifferentiation due to stable transfection with shRNA against CDH1 than against the parental cells. We evaluated whether salinomycin could also exhibit a similar activity against stem cell-like cells in multiple myeloma (MM). To establish a comparative reference for such potential activity, we first tested salinomycin (0-10 uM for up to 72hrs) against a panel of 15 MM cell lines and observed IC50 values <1 uM in 10/15 cell lines tested, including >80% reduction of tumor cell viability in 6/15 cell lines tested at 0.5 uM, i.e. levels lower than the IC50 values for in vitro activity of salinomycin against breast cancer cells with (HMLE-shCDH1, IC50 ∼1 uM) or without (HMLE-shControl, IC50 >>10 uM) stem cell-like features. CD138+ purified primary tumor cells from 3 MM patients responded to salinomycin with IC50 values (105, 332 and 750 nM, respectively) in the same range as MM cell lines. In vitro combinations with bortezomib, doxorubicin, melphalan, and dexamethasone showed overall no antagonism, while evidence of additive or even synergistic effect could be identified in certain dose ranges. Because MM cell lines and primary tumor cells responded concordantly to salinomycin and with higher sensitivity than breast cancer stem cell-like cells, we hypothesized that MM cells may in general be more responsive to salinomycin than other tumors. Since tumor-stromal interactions can increase the expression of transcriptional signatures of “stemness” in MM cells, we embarked on characterizing the anti-MM properties of salinomycin using compartment-specific bioluminescence imaging (CSBLI) assays. These showed that co-culture with stromal cells did not confer resistance to salinomycin in 5 MM cell lines (MM.1S, OCI-My5, KMS-11, KMS-18, NCI-H929) and in fact enhanced its activity against 4 of them. Side population (SP) cells, defined by their ability to efflux Hoechst stain, represent a stem cell-like population which was identified in MM cell lines and could represent the functional equivalent of the mesenchymally transdifferentiated breast cancer stem cell-like cells. We observed that salinomycin reduces the SP fraction of MM cell lines at doses >20 times lower than those required for in vitro effect against the bulk <<main population>> of the respective cell lines. Interestingly, the anti-SP effect of salinomycin was more pronounced in the presence of stroma, similarly to the CSBLI studies on the entire MM cell population and consistent with our prior observation that tumor-stroma interaction enhances transcriptional signatures of ≪stemness≫ in the tumor compartment. However, when we tested the in vivo anti-MM activity of salinomycin in an orthotopic model of i.v. injected Luc+ MM cells, no anti-MM activity (in terms of tumor burden decrease or overall survival prolongation) was observed at the maximum tolerated dose (1 mg/kg i.p. daily, which is consistent with most studies reported thus far in the literature). Ex vivo treatment of KMS-11 cells with salinomycin doses (100 nM for 72 hrs) selectively targeting SP cells was followed by s.c. injection of these cells or vehicle-treated controls in sublethallly irradiated SCID/NOD mice, but no statistically significant improvement in tumor burden or overall survival was observed. Our in vitro results indicate that salinomycin exhibits intriguing in vitro anti-MM activity, not only against SP cells but also against the bulk ≪main≫ MM cell population, even in the presence of stromal support. In contrast, the in vivo activity of salinomycin is compromised by side effects in the orthotopic model of MM lesions, while short term ex vivo exposure of tumor cells is conceivably insufficient to eradicate clonogenic cells and lead to appreciable delay in tumor growth in vivo. Our studies point to intriguing features as well as notable challenges that have to overcome before salinomycin or other more selective agents of this class can be safely tested in clinical trials in MM. Disclosures: McMillin: Axios Biosciences: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Birinapant (TL32711), a Small Molecule Smac Mimetic, Induces Regressions in Childhood Acute Lymphoblastic Leukemia (ALL) Xenografts That Express TNFα and Synergizes with TNFα in Vitro – A Report From the Pediatric Preclinical Testing Program (PPTP)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3565-3565 ◽  
Author(s):  
Malcolm A. Smith ◽  
Hernan Carol ◽  
Kathryn Evans ◽  
Jennifer Richmond ◽  
Min Kang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Solid Tumor ◽  
Single Agent ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Tumor Xenografts ◽  
Normal Tissues ◽  
Smac Mimetics

Abstract Abstract 3565 Introduction: Birinapant is a small molecule mimetic of Smac that potently and specifically antagonizes multiple inhibitors of apoptosis proteins (IAPs). Birinapant rapidly degrades cIAPs and enables cytokines (TNFα, TRAIL) to activate the extrinsic apoptosis pathway, while it rapidly turns off the canonical NF-κB survival pathway, causing cancer cell death. Preclinical studies using adult cancer models have shown that birinapant causes tumor regressions as a single agent in selected models and that it has potent antitumor activity when combined with chemotherapies and death receptor ligands. Methods: Birinapant was evaluated against the 23 cell lines of the PPTP in vitro panel (including 1 AML and 5 ALL lines) using 96 hour exposure at concentrations from 1.0 nM to 3.0 μM, both as a single agent and in combination with TNFα (10 ng/mL) or TRAIL (10 ng/mL). Birinapant was tested against 2 PPTP solid tumor xenografts (rhabdomyosarcoma, Rh30; Ewing, CHLA-258), an anaplastic large cell lymphoma (ALCL) xenograft (Karpas-299), and 3 B-precursor ALL xenografts (ALL-2, ALL-17, & ALL-19) at a dose of 30 mg/kg administered by the intraperitoneal route using a Q3 day × 5 schedule. Gene expression data for the PPTP cell lines and xenografts was available using both Affymetrix U133 Plus 2.0 and Agilent SurePrint G3 arrays. Results: Birinapant demonstrated limited single agent activity (median relative IC50 (rIC50) > 3 μM), with only the AML cell line Kasumi-1 showing Relative In/Out% (Rel I/O%) values < 0% with rIC50 of 37 nM. Marked potentiation of birinapant was observed for a subset of cell lines with the addition of TNFα or TRAIL. The 5 ALL cell lines showed a median rIC50 value of 3.6 nM for birinapant in combination with TNFα, with Rel I/O% values between −50% and −100% (indicative of a profound cytotoxic effect). Four of 5 ALL cell lines showed little or no potentiation of birinapant effect with the addition of TRAIL. Among solid tumor cell lines, potentiation of birinapant effect was observed for selected rhabdomyosarcoma, rhabdoid tumor, Ewing sarcoma, and neuroblastoma cell lines with the addition of either TNFα or TRAIL. Birinapant was well tolerated in vivo. Birinapant induced significant differences in event-free survival (EFS) distribution compared to control in 3 of 3 (100%) of the B-precursor ALL xenografts, but in none of the solid tumor or ALCL xenografts. Objective responses were not observed for the solid tumor and ALCL xenografts, whereas for the ALL panel one xenograft (ALL-17) achieved a complete response (CR) and another (ALL-2) achieved a maintained CR, with treated animals remaining in remission at day 42, approximately 30 days after their last treatment with birinapant. Given the mechanism of action of Smac mimetics, TNFα expression was examined. TNFα expression was significantly higher for the PPTP ALL xenografts compared to the PPTP solid tumor xenografts and to 15 normal tissues. TNFα expression in ALL clinical specimens was examined using publicly available datasets, with the observation that its expression is significantly higher for high-risk B-precursor ALL compared to a set of normal tissues, but with a wide range of TNFα expression among ALL cases. Lymphotoxin A and B also show significantly elevated expression in ALL compared to normal tissues. Among the ALL xenografts tested with birinapant, the best responding xenograft (ALL-2) showed the highest TNFα expression. Karpas-299 also showed high TNFα expression, but the two solid tumor xenografts did not. Unlike the ALL cell lines for which exogenous TNFα potentiated birinapant in vitro activity, exogenous TNFα did not potentiate birinapant in vitro activity against Karpas-299. Conclusion: Birinapant showed little single agent in vitro activity against ALL cell lines, though it markedly potentiated the activity of exogenously added TNFα for these cell lines. In vivo, birinapant showed remission-inducing activity against 2 of 3 ALL xenografts, with one of these showing a maintained CR. TNFα is mechanistically associated with the activity of Smac mimetics, and the initial PPTP in vivo data for ALL xenografts are consistent with a relationship between TNFα expression and responsiveness to birinapant. The PPTP results suggest that birinapant may show high level activity against a subset of childhood ALL, and additional in vivo testing is ongoing to better identify predictive markers that can reliably select responsive cases. Disclosures: Chunduru: TetraLogic Pharmaceuticals: Employment, Equity Ownership. Graham:TetraLogic Pharmaceuticals: Employment, Equity Ownership. Geier:TetraLogic Pharmaceuticals: Honoraria. Houghton:TetraLogic Pharmaceuticals: Honoraria.

scholarly journals NK cells promote cancer immunoediting through tumor-intrinsic loss of interferon-stimulated gene expression

2020 ◽  
Author(s):  
Yung Yu Wong ◽  
Luke Riggan ◽  
Edgar Perez-Reyes ◽  
Christopher Huerta ◽  
Matt Moldenhauer ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Metastatic Cancer ◽  
Cancer Immunoediting ◽  
Ifn Γ

AbstractNatural killer (NK) cells are innate lymphocytes that constantly patrol host tissues against transformed cells in a process known as cancer immunosurveillance. Previous evidence in mice has demonstrated that NK cell-derived IFN-γ can promote immunoevasion by sculpting the immunogenicity of developing tumors in a process known as cancer immunoediting. This process involves the elimination of highly immunogenic “unedited” tumor cells followed by the eventual escape of less immunogenic “edited” tumor cell variants that are able to escape recognition or elimination by the immune system. Here, we show that NK cell-edited fibrosarcomas decrease the expression of 17 conserved IFN-γ-inducible genes compared to unedited tumor cells. High expression of 3 of these identified genes (Psmb8, Trim21, Parp12) in human tumor samples correlates with enhanced survival in breast cancer, melanoma, and sarcoma patients. While NK cell-edited fibrosarcomas displayed resistance to IFN-γ growth suppression in vitro, functional knockouts of individual interferon stimulated genes (ISGs) were not required for outgrowth of unedited tumor cell lines in vitro and in vivo compared to complete loss of IFN signaling. Furthermore, knockout of IFN-γ-intrinsic signaling via deletion of Ifngr in edited B16 F10 and E0771 LMB metastatic cancer cell lines did not impact host survival following lung metastasis. Together, these results suggest that unedited tumors can be selected for decreased IFN-γ signaling to evade immune responses in vivo, and as a consequence, tumor-extrinsic IFN signaling may be more important for potentiating durable anti-tumor responses to advanced solid tumors.

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