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.

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.

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.

In Vitro Effect of Focused Ultrasound or Thermal Stress on HSP70 Expression and Cell Viability in Three Tumor Cell Lines

2007 ◽  
Vol 14 (7) ◽  
pp. 859-870 ◽  
Author(s):  
Walter Hundt ◽  
Caitlin E. O’Connell-Rodwell ◽  
Mark D. Bednarski ◽  
Silke Steinbach ◽  
Samira Guccione
Keyword(s):  
Thermal Stress ◽  
Tumor Cell ◽  
Cell Viability ◽  
Cell Lines ◽  
Focused Ultrasound ◽  
Hsp70 Expression ◽  
Tumor Cell Lines ◽  
Vitro Effect

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.

Anti-Myeloma Effects of the Janus Kinase 2 (JAK2) Inhibitor SAR503 Alone and in Combination Treatment

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4986-4986
Author(s):  
Haiming Chen ◽  
Mingjie Li ◽  
Jennifer Li ◽  
Kevin Delijani ◽  
Danielle Rauch ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Cell ◽  
Cell Viability ◽  
Tumor Cells ◽  
Cell Lines ◽  
Drug Exposure ◽  
Single Agent ◽  
Janus Kinase ◽  
Gm Csf ◽  
Jak2 Inhibitor

Abstract Abstract 4986 Background: Janus kinase 2 (JAK2) is a cytoplasmic tyrosine kinase that carries out a series of cascading signals via signal transducer and activator of transcription (STAT)s, mitogen-activated protein kinase (MAPK), and phosphorylation of PI3K. Activation of the JAK2 pathway plays an important role in both normal and malignant hematopoiesis. The JAK pathway ha been shown to play a key role in multiple myeloma (MM). JAK2 has been specifically implicated in signaling by members of the type II cytokine receptor family (interferon [IFN] receptor), GM-CSF receptor (IL-3R, IL-5R, and GM-CSF-R), gp130 receptor family interleukin-6 (IL-6R) and single chain receptors (Epo-R, Tpo-R, GH-R, and PRL-R). IFN-α inhibits MM cell proliferation in association with cell cycle arrest at G1 and limits the clonogenic growth of both MM cell lines and primary MM patient specimens. SAR503 (Sanofi-Aventis) is a potent, highly selective JAK2 inhibitor. Thus, we evaluated the anti-MM effects of SAR503 as a single agent and in combination with other anti-MM drugs and evaluated gene and protein expression in MM cells exposed to these drugs. Experiment design: The MM cell lines RPMI8226, U266, and MM1s were cultured in RPMI1640 with standard nutrition supplements. Bone marrow aspirates were obtained from MM patients following informed consent. Bone marrow mononuclear cells (BMMCs) were isolated by using density-gradient centrifugation with Histopaque-1077 (Sigma, St Louis). Cells were plated in 96 well plates at a concentration of 6 × 104 cells/100 ml/well, and incubated for 24 hours prior to drug treatment, after which time the drugs were added in replicates of six for 48 hours. BMMCs were incubated in the presence of media, SAR503, doxorubicin, melphalan, dexamethasone, bortezomib, or IFN-α alone or the combination of SAR503 with one of these anti-MM agents. Following the 48-hour drug incubation, cell viability was assessed utilizing the cell proliferation MTS assay. For gene expression studies, total RNA was isolated MM tumor cells with or without drug exposure. RNA was reverse-transcribed into cDNA and amplified using the Thermo-Script RT-PCR System and PCR performed again using the GeneAmp PCR System 9700. Protein phosphorylation of MM tumor cells with or without drug exposure was determined with Western blot analysis. Results: SAR503 alone inhibited MM tumor cell proliferation in a concentration-dependent fashion. The 50% growth inhibition (IC50) of cells from MM cell lines at 48 hours varied (IC50: RPMI8226 1mM; U266 0. 5mM; MM1s 10mM). IC50 of primary MM tumor cells treated with SAR503 ranged from approximately 5 to 10mM in different patients. Notably, the combination of SAR503 and either doxorubicin or melphalan showed markedly reduced cell viability compared to either drug alone in all three MM cell lines and primary tumor cells from MM patients. Since this effect may have resulted from decreased cell proliferation due to inhibition of the JAK2 pathway and cell cycle arrest or increased cell death, we further determined cell apoptosis of MM tumor cells treated with SAR503 alone by using flow cytometric analysis to detect Annexin V and propidium iodide (PI) staining. Our data showed SAR503 increased MM tumor cell apoptosis in a concentration-dependent fashion. The combination of SAR503 and dexamethasone or bortezomib only slightly reduced tumor cell viability in both MM cell lines and primary MM tumor cells more than single agent treatment, and the combination of SAR503 with IFN-α did not enhance the anti-MM effects compared to single drug treatment. Notably, RT-PCR results showed marked decreases in both AKT1 and mTOR gene expression in MM tumor cells treated with SAR503. Conclusion: The combination of the JAK2 inhibitor SAR503 with doxorubicin or melphalan markedly reduces MM tumor cell viability more than single agent treatment. The results from these studies suggest that enhanced anti-MM activity may be observed when SAR503 is combined with conventional treatment for MM. We are currently evaluating the anti-MM effects of SAR503 in these combination treatments in vivo using our MM xenograft models. Disclosures: Berenson: Onyx: Consultancy, Honoraria, Speakers Bureau.

scholarly journals Combined Targeting of Distinct c-Myc and JunB Transcriptional Programs Inducing Synergistic Anti-Myeloma Activity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2644-2644
Author(s):  
Judith Lind ◽  
Sonia Vallet ◽  
Karoline Kollmann ◽  
Osman Aksoy ◽  
Vincent Sunder-Plassmann ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Cell ◽  
Cell Lines ◽  
Ex Vivo ◽  
Mapk Signaling ◽  
Loss Of Function ◽  
Tumor Cell Death ◽  
Expression Levels ◽  
Protein Levels

Abstract INTRODUCTION Transcription factors (TFs) are convergence points of signaling cascades that coordinate cell differentiation, proliferation and survival and are commonly deregulated in cancer, including multiple myeloma (MM). They contribute to the initiation of MM and promote tumor cell growth and drug resistance. Both cMyc, a merging point of the PI3K-, and JunB, a merging point of the MEK/MAPK-signaling pathway, play pivotal roles in MM. Exciting novel approaches to inhibit TFs like proteolysis-targeting-chimera (PROTAC) promise to lead to selective tumor cell death with little/no consequence for normal cells. However, redundancy phenomena of transcriptional programs are likely to challenge their efficacy. Here, we report our final results on combined targeting of distinct c-Myc & JunB transcriptional programs for MM therapy. METHODS MM cell lines and patient MM cells were analyzed. Following CRISPR-loss-of-function screens for cMyc & JunB across MM cell lines and correlation analyses in MM patient datasets, the functional relevance of BRD4/c-Myc- and MEK/JunB-induced TF programs was delineated using genomic and chemical approaches in 2D and 3D models of the bone marrow (BM) microenvironment. Specifically, effects of single or combined targeting of cMyc- and JunB-induced TF-programs were analyzed by flow cytometry, western blot, RNAseq, qPCR and luciferase assays. In vitro and ex vivo results were finally verified in a MM xenograft mouse model. RESULTS While CRISPR loss-of-function screens across various MM cell lines confirmed their growth dependency on cMyc and JunB, we did not observe correlative expression levels among these TFs, neither in the publicly available GSE6477 nor in the CoMMpass dataset. In contrast, a significant positive correlation was observed between Brd4 and cMyc, and MEK and JunB expression levels, respectively. The existence of two distinct Brd4/cMyc and MEK/JunB transcriptional programs in MM cells was subsequently supported by a lack of changes in cMyc mRNA/protein levels and resultant transcriptional activity upon JunB knockdown, and vice versa. Likewise, MZ-1, a novel PROTAC which targets Brd4, resulted in the inhibition of BMSC/IL-6- induced cMyc- but not JunB- upregulation. Conversely, neither the MEK inhibitor trametinib nor doxycycline-induced knockdown of BMSC/IL-6- induced JunB upregulation in TetshJunB/MM.1S cells reduced Brd4/c-Myc mRNA/protein levels. Importantly, the activity of MZ-1 and trametinib was predicted by Brd4 and JunB expression levels using mathematical models, respectively. Further, combination of MZ-1 with trametinib or JunB knockdown synergistically inhibited tumor cell proliferation, and induced cell death in a 2D and a dynamic 3D model of the MM-BM milieu. Finally, our in vitro and ex vivo results were confirmed in vivo, utilizing BMSC:TetshJunB/MM.1S vs. BMSC:TetshSCR/MM.1S-carrying NSG mice treated with MZ-1 with/without doxycycline or trametinib. CONCLUSION In summary, our data demonstrate for the first time the existence of non-overlapping cMyc and JunB-regulated TF programs providing a rationale for combined cMyc:JunB targeting treatment strategies in MM. Disclosures Vallet: Pfizer: Honoraria; MSD: Honoraria; Roche Pharmaceuticals: Consultancy. Podar: Celgene: Consultancy, Honoraria; Roche Pharmaceuticals: Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria; Amgen Inc.: Consultancy, Honoraria.

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.

scholarly journals The Sumoylation Inhibitor TAK-981 in Combination with the CD19-Targeting Antibody Tafasitamab Shows Enhanced Anti-Tumor Activity in Preclinical B-Cell Lymphoma Models

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2268-2268
Author(s):  
Maria Patra-Kneuer ◽  
Akito Nakamura ◽  
Keli Song ◽  
Stephen Grossman ◽  
Andrea Polzer ◽  
...  
Keyword(s):  
Cell Viability ◽  
Tumor Cells ◽  
Cell Lines ◽  
Burkitt Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Effector Cells ◽  
Xenograft Models ◽  
Development Center

Abstract Introduction TAK-981 is a first-in-class small molecule inhibitor of the SUMO activating enzyme currently in Phase I/II clinical trials. TAK-981 has been shown to increase NK cell activation and M1 macrophage polarization via upregulation of Type I interferon (IFN) signaling, leading to enhanced antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in combination with rituximab (Nakamura 2019, AACR). Tafasitamab (MOR208) is a CD19-targeting antibody with enhanced Fc effector function mediating ADCC, ADCP and direct cytotoxic activities against B-lymphoma cells. Based on the Phase II clinical study L-MIND (Salles et al., 2020 and Duell et al., 2021), tafasitamab in combination with lenalidomide received accelerated approval by the Food and Drug Administration for the treatment of transplant-ineligible adult patients with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL). Due to the potential for TAK-981 to enhance the activity of tafasitamab via activation of innate effector cells, we aimed to investigate the effects of this drug combination on ADCC, ADCP and tumor cell viability in vitro. Additionally, combinatorial activity of TAK-981 plus tafasitamab was evaluated in lymphoma xenograft models. Methods A panel of 9 aggressive lymphoma cell lines was analyzed (7 DLBCL and 2 Burkitt lymphoma). For ADCC, PBMC effector cells from healthy human donors were pre-treated with 0.1 or 1 µM TAK-981 or dimethyl sulfoxide (DMSO) control for 24 hours. Tumor cells were incubated with/without 1 nM tafasitamab in the presence of TAK-981 pretreated PBMCs at effector-to-target (E:T) ratios of 5:1 to 10:1 for 2 hours. Degranulation of NK cells was determined via CD107a surface expression after co-incubation of TAK-981 pre-treated PBMCs with tumor cells and 0.1 or 10 nM tafasitamab for 3 hours. Cytokine levels in the supernatant were investigated upon incubation of PBMCs with lymphoma cells, 1 µM TAK-981 and/or 10 nM tafasitamab for 24 hours. For the ADCP assays, in vitro differentiated macrophages were treated with 1 µM TAK-981 for 24 hours. Next, macrophages were incubated with lymphoma cells and 1 or 10 nM tafasitamab at an E:T ratio of 2:1 for 3 hours. For cell viability assays, tumor cells were treated with 1-1000 nM TAK-981 and/or 5 nM tafasitamab for 24 hours in the absence of effector cells. Cytotoxicity, phagocytosis, degranulation and cytokine release were analyzed by flow cytometry. Cell viability was assessed by determination of ATP levels. For in vivo analysis, effects of TAK-981 (7.5 mg/kg IV twice weekly) in combination with tafasitamab (3, 10 or 20 mg/kg IP twice weekly) on tumor growth were evaluated in Daudi and WSU-DLCL2 xenograft models of Burkitt lymphoma and DLBCL grown in SCID mice. Results In ADCC experiments, increased cytotoxicity was observed upon combination treatment with TAK-981 and tafasitamab compared to the respective mono treatments in 5/8 tested lymphoma cell lines (Daudi, SU-DHL-2, SU-DHL-6, TMD8, OCI-LY10). Moreover, TAK-981 plus tafasitamab enhanced degranulation of NK cells and cytokine release compared to mono treatments. In ADCP assays, combination of TAK-981 and tafasitamab resulted in increased phagocytosis rates in comparison to mono treatments in 2/2 tested cell lines (Daudi, Ramos). Cell viability analysis revealed a combination benefit by increased direct cytotoxic effects against SU-DHL-6 cells. Finally, TAK-981 and tafasitamab were investigated in Daudi and WSU-DLCL2 xenograft models with 3 weeks of dosing. In the Daudi model, the combination treatments of TAK-981 with 10 or 20 mg/kg tafasitamab performed better than either treatment alone, and in the WSU-DLCL2 model, the combination treatments of TAK-981 with 3, 10 or 20 mg/kg tafasitamab performed better than the single agent treatments. Conclusions The combination of TAK-981 with tafasitamab significantly enhanced anti-tumor effects compared to the respective monotherapies in vitro and in vivo. These preclinical data support a clinical evaluation of this drug combination in patients with lymphoma including aggressive subtypes such as Burkitt lymphoma and DLBCL. The study was funded by MorphoSys AG and Takeda Development Center Americas, Inc. Disclosures Patra-Kneuer: MorphoSys AG: Current Employment. Nakamura: Takeda Development Center Americas, Inc.: Current Employment. Song: Takeda Pharmaceuticals International Co.: Current Employment. Grossman: Takeda Development Center, Cambridge MA: Current Employment. Polzer: MorphoSys: Current Employment. Ginzel: MorphoSys: Current Employment. Steidl: MorphoSys AG: Current Employment. Berger: Takeda Development Center Americas, Inc.: Current Employment. Proscurshim: Takeda Pharmaceuticals: Current Employment, Current holder of individual stocks in a privately-held company. Heitmüller: MorphoSys AG: Current Employment.

scholarly journals Actin-binding protein profilin1 promotes aggressiveness of clear-cell renal cell carcinoma cells

2020 ◽  
Vol 295 (46) ◽  
pp. 15636-15649 ◽  
Author(s):  
Abigail Allen ◽  
David Gau ◽  
Paul Francoeur ◽  
Jordan Sturm ◽  
Yue Wang ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Tumor Cell ◽  
Stromal Cells ◽  
Clear Cell ◽  
Actin Binding ◽  
Vascular Endothelial ◽  
Cell Renal Cell Carcinoma ◽  
And Migration ◽  
Proliferation And Migration

Clear-cell renal cell carcinoma (ccRCC), the most common subtype of renal cancer, has a poor clinical outcome. A hallmark of ccRCC is genetic loss-of-function of VHL (von Hippel–Lindau) that leads to a highly vascularized tumor microenvironment. Although many ccRCC patients initially respond to antiangiogenic therapies, virtually all develop progressive, drug-refractory disease. Given the role of dysregulated expressions of cytoskeletal and cytoskeleton-regulatory proteins in tumor progression, we performed analyses of The Cancer Genome Atlas (TCGA) transcriptome data for different classes of actin-binding proteins to demonstrate that increased mRNA expression of profilin1 (Pfn1), Arp3, cofilin1, Ena/VASP, and CapZ, is an indicator of poor prognosis in ccRCC. Focusing further on Pfn1, we performed immunohistochemistry-based classification of Pfn1 staining in tissue microarrays, which indicated Pfn1 positivity in both tumor and stromal cells; however, the vast majority of ccRCC tumors tend to be Pfn1-positive selectively in stromal cells only. This finding is further supported by evidence for dramatic transcriptional up-regulation of Pfn1 in tumor-associated vascular endothelial cells in the clinical specimens of ccRCC. In vitro studies support the importance of Pfn1 in proliferation and migration of RCC cells and in soluble Pfn1's involvement in vascular endothelial cell tumor cell cross-talk. Furthermore, proof-of-concept studies demonstrate that treatment with a novel computationally designed Pfn1–actin interaction inhibitor identified herein reduces proliferation and migration of RCC cells in vitro and RCC tumor growth in vivo. Based on these findings, we propose a potentiating role for Pfn1 in promoting tumor cell aggressiveness in the setting of ccRCC.

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