Stem-like Cell Lines Research Articles

Abstract LB-175: ATM regulates radiation-induced translational control of gene expression

Abstract The cellular radioresponse is composed of processes regulated by constitutively expressed proteins and processes regulated by new gene expression. While processes regulated by constitutively expressed proteins, such as those participating in DNA repair and cell cycle arrest, have been extensively studied, the radiation-induced changes in gene expression are not well defined. Previously, we have demonstrated that translation plays an important role in regulating gene expression in response to radiation. In glioblastoma cells, treatment with radiation affects more genes at the translational level than at the transcriptional level. Specific transcripts were found to be recruited to or away from polysomes without global changes in the polysome profile of the cells. Here, we utilized microarray analysis of polysome-associated transcripts in the glioblastoma stem-like cell line NSC11 to demonstrate that the DNA damage response kinase ATM controls the translation of a subset of genes as part of the radioresponse. Cells were incubated with the ATM inhibitor KU-60019 at a dose of 500nM for one hour, exposed to 2 Gy, and harvested for polysome extraction one hour after irradiation. ATM inhibition blocked translational changes of approximately one third of genes that were affected by radiation. Gene enrichment analysis revealed that genes translationally regulated by ATM were significantly involved in a number of cellular processes, indicating that ATM-mediated translational control may have functional consequences on the cellular radioresponse. By GSEA, cells treated with radiation alone were highly enriched for terms involving mitochondrial structure and function, cellular respiration, and oxidative stress as compared to irradiated cells treated with the ATM inhibitor. Other significantly enriched categories of functional interest included the spliceosome, RNA catabolic processes, the small nuclear ribonucleoprotein complex, and phospholipid and phosphoinositide metabolism. Analysis of UTR regulatory elements revealed that a significant percentage of the genes translationally upregulated by ATM in irradiated cells were enriched for HuR binding sites, which suggests a potential mechanism for polysome recruitment of these transcripts. Overall, these findings uncover a novel network of post-transcriptional gene regulation orchestrated by ATM and provide a framework to identify potential new molecules and pathways that govern tumor cell radiosensitivity. Citation Format: Stacey L. Lehman, Amy Wahba, Kevin Camphausen, Philip J. Tofilon. ATM regulates radiation-induced translational control of gene expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-175.

Abstract 3235: IDH mutant gliomas escape natural killer cell immune surveillance by downregulation of NKG2D ligands

Abstract Background: Diffuse gliomas are fatal primary brain tumors that are poorly immunogenic. The basis for insufficient anti-tumor immunity in diffuse gliomas is not understood. Gain of function mutations in isocitrate dehydrogenases (IDH1 and IDH2) promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Here, we identify epigenetic dysregulation of natural killer (NK) cell ligand genes as significant contributors to immune escape in glioma. Methods: We analyzed the TCGA database for immune gene expression patterns in IDH mutant or wild-type gliomas and identified differentially expressed immune genes. NKG2D ligands expression levels and NK cell-mediated lysis were measured in IDH mutant and wild-type patient-derived glioma stem cells and in genetically engineered astrocytes. Finally, we assessed the impact of hypomethylating agent 5-aza- 2’deoxycytodine (Decitabine) as a potential NK cell sensitizing agent in IDH mutant cells. Results: All IDH mutant glioma stem-like cell lines exhibited significantly lower expression of NKG2D ligands compared with IDH wild-type cells. Consistent with these findings, IDH mutant glioma cells and astrocytes were resistant to NK cell- mediated lysis. The hypomethylating agent Decitabine increased NKG2D ligand expression, and restored NK-mediated lysis of IDH mutant cells in an NKG2D- dependent manner. Conclusions: IDH mutant glioma cells acquire resistance to NK cells through epigenetic silencing of NKG2D ligands ULBP1 and ULPB3. Decitabine-mediated hypomethylation restores ULBP1 and ULBP3 expression in IDH mutant glioma cells and may provide a clinically useful method to sensitize IDH mutant gliomas to NK cell- mediated immune surveillance in patients with IDH mutated diffuse gliomas. Citation Format: Aparna Rao, Xiaoran Zhang, Christopher Deibert, Paola Sette, Tim Chan, Paola Grandi, Nduka Amankulor. IDH mutant gliomas escape natural killer cell immune surveillance by downregulation of NKG2D ligands. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3235.

Polysome Profiling Links Translational Control to the Radioresponse of Glioblastoma Stem-like Cells.

Changes in polysome-bound mRNA (translatome) are correlated closely with changes in the proteome in cells. Therefore, to better understand the processes mediating the response of glioblastoma to ionizing radiation (IR), we used polysome profiling to define the IR-induced translatomes of a set of human glioblastoma stem-like cell (GSC) lines. Although cell line specificity accounted for the largest proportion of genes within each translatome, there were also genes that were common to the GSC lines. In particular, analyses of the IR-induced common translatome identified components of the DNA damage response, consistent with a role for the translational control of gene expression in cellular radioresponse. Moreover, translatome analyses suggested that IR enhanced cap-dependent translation processes, an effect corroborated by the finding of increased eIF4F-cap complex formation detected after irradiation in all GSC lines. Translatome analyses also predicted that Golgi function was affected by IR. Accordingly, Golgi dispersal was detected after irradiation of each of the GSC lines. In addition to the common responses seen, translatome analyses predicted cell line-specific changes in mitochondria, as substantiated by changes in mitochondrial mass and DNA content. Together, these results suggest that analysis of radiation-induced translatomes can provide new molecular insights concerning the radiation response of cancer cells. More specifically, they suggest that the translational control of gene expression may provide a source of molecular targets for glioblastoma radiosensitization. Cancer Res; 76(10); 3078-87. ©2016 AACR.

IDH Mutant Gliomas Escape Natural Killer Cell Immune Surveillance by Downregulation of NKG2D Ligand Expression

Abstract Background Diffuse gliomas are fatal primary brain tumors that are poorly immunogenic. The basis for insufficient anti-tumor immunity in diffuse gliomas is not understood. Mutations in isocitrate dehydrogenases (IDH1 and IDH2) promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Here, we identify epigenetic dysregulation of natural killer (NK) cell ligand genes as significant contributors to immune escape in glioma. Methods We analyzed the TCGA database for immune gene expression patterns in IDH mutant or wild-type gliomas and identified differentially expressed immune genes. NKG2D ligands expression levels and NK cell-mediated lysis were measured in patient-derived glioma stem cells and in genetically engineered astrocytes. Finally, we assessed the impact of hypomethylating agent Decitabine as a potential NK cell sensitizing agent in IDH mutant cells. Results All IDH mutant glioma stem-like cell lines exhibited significantly lower expression of NKG2D ligands compared with IDH wild-type cells. Consistent with these findings, IDH mutant glioma cells and astrocytes were resistant to NK cell-mediated lysis. The hypomethylating agent Decitabine increased NKG2D ligand expression, and restored NK- mediated lysis of IDH mutant cells in an NKG2D-dependent manner. Conclusions IDH mutant glioma cells acquire resistance to NK cells through epigenetic silencing of NKG2D ligands ULBP1 and ULPB3. Decitabine-mediated hypomethylation restores ULBP1 and ULBP3 expression in IDH mutant glioma cells and may provide a clinically useful method to sensitize IDH mutant gliomas to NK cell-mediated immune surveillance in patients with IDH mutated diffuse gliomas.

IDH mutant gliomas escape natural killer cell immune surveillance by downregulation of NKG2D ligand expression.

Diffuse gliomas are poorly immunogenic, fatal brain tumors. The basis for insufficient antitumor immunity in diffuse gliomas is unknown. Gain-of-function mutations in isocitrate dehydrogenases (IDH1 and IDH2) promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Here, we identify epigenetic dysregulation of natural killer (NK) cell ligand genes as significant contributors to immune escape in glioma. We analyzed the database of The Cancer Genome Atlas for immune gene expression patterns in IDH mutant or wild-type gliomas and identified differentially expressed immune genes. NKG2D ligand expression levels and NK cell-mediated lysis were measured in IDH mutant and wild-type patient-derived glioma stem cells and genetically engineered astrocytes. Finally, we assessed the impact of hypomethylating agent 5-aza-2'deoxycytodine (decitabine) as a potential NK cell sensitizing agent in IDH mutant cells. IDH mutant glioma stemlike cell lines exhibited significantly lower expression of NKG2D ligands compared with IDH wild-type cells. Consistent with these findings, IDH mutant glioma cells and astrocytes are resistant to NK cell-mediated lysis. Decitabine increases NKG2D ligand expression and restores NK-mediated lysis of IDH mutant cells in an NKG2D-dependent manner. IDH mutant glioma cells acquire resistance to NK cells through epigenetic silencing of NKG2D ligands ULBP1 and ULBP3. Decitabine-mediated hypomethylation restores ULBP1 and ULBP3 expression in IDH mutant glioma cells and may provide a clinically useful method to sensitize IDH mutant gliomas to NK cell-mediated immune surveillance in patients with IDH mutated diffuse gliomas.

PD35-09 DIFFERENTIAL ACTIONS OF ERα AND ERβ VIA NON-GENOMIC SIGNALING IN HUMAN PROSTATE STEM-PROGENITOR CELLS

You have accessJournal of UrologyStem Cell Research1 Apr 2016PD35-09 DIFFERENTIAL ACTIONS OF ERα AND ERβ VIA NON-GENOMIC SIGNALING IN HUMAN PROSTATE STEM-PROGENITOR CELLS Shyama Majumdar, Neha Malhotra, Susan Kasper, Lishi Xie, Timothy Gauntner, Wen-yang Hu, and Gail Prins Shyama MajumdarShyama Majumdar More articles by this author , Neha MalhotraNeha Malhotra More articles by this author , Susan KasperSusan Kasper More articles by this author , Lishi XieLishi Xie More articles by this author , Timothy GauntnerTimothy Gauntner More articles by this author , Wen-yang HuWen-yang Hu More articles by this author , and Gail PrinsGail Prins More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1081AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Estrogens have been implicated in prostate development and cancer; however, specific roles of estrogen receptor-α and estrogen receptor-β are not well established. Using human prostate stem-progenitor cells we have previously shown that non-genomic pathways are involved in estrogen actions. Our present study sought to elucidate specific roles for ERα and ERβ via membrane initiated signaling in stem-progenitor cells. METHODS Human prostate stem-progenitor cells were enriched from primary prostate epithelial cell culture of young, disease-free donors using a 3D prostasphere (PS) model as previously described. Cells were labeled using ERα or ERβ antibodies and prostate stem cell markers CD49f and TROP2 followed by triple channel FACS to quantify ERα+/ERβ+ cell numbers. To explore ERα, the benign human prostate stem cell line WPE-stem, with extremely low levels of ERα and ERβ, was stably transfected with a lentiviral-ERα expression vector. The human prostate cancer stem-like cell line HuSLC (ERβ++, ERα-) was used to interrogate ERβ actions. Cells were exposed to 10 nM estradiol (E2) over a 15 to 60 minute time course +/- ICI 182,870 (ICI), an ERα/β antagonist. Western Blotting was used to quantify phosphorylation of target proteins. RESULTS FACS analysis of day 7 PS cells labeled for ERα or ERβ revealed 66% of day 7 PS cells as ERα+ and 40% as ERβ+. Among ERα or ERβ positive PS cells, 4% were Trop2+/CD49fhigh (stem-like cells) and 10-12% were Trop2+/CD49fmedium (early stage progenitor cells). PS exposed to 10 nM E2 showed sequential phosphorylation of Src, Erk1/2, p38, Akt and NFκB (p65) over 60 minutes. ICI attenuated Akt and Erk1/2 phosphorylation, confirming membrane bound ERs are involved in downstream signaling. E2 treatment of HuSLCs showed phosphorylation of Erk1/2 but not Akt, indicating that ERβ signals exclusively through the MAPK pathway in these cells. Conversely, E2 treatment of WPE-stem cells overexpressing ERα resulted in robust phosphorylation of Akt but lower levels of Erk1/2 phosphorylation suggesting that Akt activation may be more reliant on ERα signaling. CONCLUSIONS ERs trigger cellular responses via activation of signaling cascades that originate at the membrane (non-genomic signaling) in addition to nuclear signaling. The present findings reveal that human prostate stem-progenitor cells express both ERα and ERβ, which differentially activate membrane signaling cascades. This may lead to unique downstream actions that influence prostate stem-progenitor cell proliferation as well as lineage commitment decisions. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e847 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Shyama Majumdar More articles by this author Neha Malhotra More articles by this author Susan Kasper More articles by this author Lishi Xie More articles by this author Timothy Gauntner More articles by this author Wen-yang Hu More articles by this author Gail Prins More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract B41: Withaferin A promotes ROS-mediated differentiation of neuroblastoma

Abstract Background: Neuroblastoma (NB), the most common extra-cranial solid tumor in children, originates from the precursor neuroblasts of the sympathetic nervous system. NB accounts for approximately 7-10% of childhood cancers and 15% of childhood cancer death. Despite an aggressive treatment regimen, the 5-year survival for high risk NB remains less than 50%. The differentiation of NB cells into mature cells represents a promising strategy for NB therapy. Currently, retinoids are the most commonly used differentiating agents. However, their use can be limited due to intrinsic or acquired resistance, as well as toxicity. We sought to evaluate the potential of the natural product withaferin A (WA), a steroidal lactone derived from the medicinal plant Withania somnifera, to induce NB cell differentiation. Methods: For differentiation studies NB cell lines (NB1691, SMS-KCNR, SH-SY5Y and the primary cell line SVBM15) were exposed to WA (100-500nM) for 7-10 days and evaluated by light microscopy, immunocytochemistry and western blot analysis. NB stem-like cell lines were generated by culturing NB1691 and SVBM15 cells in neurosphere media. To determine the IC50 (concentration needed to reduce viability by 50%), NB stem-like cell lines were exposed to increasing concentrations of WA and viability was assessed at 72 hours using MTS assay. Reactive oxygen species (ROS) were detected using CM-H2DFDA and ROS induction was inhibited with N-acetyl-L- Cysteine (NAC). To determine WA effect on neurosphere formation, NB cells were plated in 96-well plates at 50 cells/well. Cells were grown in increasing concentrations of WA and spheres were counted at 14 days. Results: WA promoted morphologic alterations (neurite outgrowth) and growth inhibition in a dose dependent manner. Immunocytochemistry and western blot analysis indicated an increase in neuronal markers including neurofilament, β-tubulin and MAP2, as well as a decrease in the stem cell markers BMi-1 and musashi. WA promoted ROS induction, which could be prevented with NAC pretreatment. NAC prevented WA-induced morphological changes and inhibited WA-induced changes in stem cell and differentiation marker expression. WA induced NB stem-like cell death in a dose dependent manner (IC50 of NB1691=1.05 μM; SVBM15=1.06 μM), and significantly inhibited neurosphere formation at concentrations as low as 50nM, which did not exhibit cytotoxicity in regular cell culture conditions. Conclusion: Withania somnifera has been used for centuries and is commonly used in ayurvedic medicine. WA has been shown to affect multiple pathways important for cancer progression and induce anti-cancer effects in breast, prostate and pancreatic cancers. Our data indicates that WA induces NB stem-like cell death, and promotes ROS-mediated NB cell differentiation. Differentiation therapy aims at reducing the risk of the tumor regrowth following high-dose chemotherapy and stem cell transplant in patients classified as high-risk. WA holds great promise as a novel alternative NB treatment strategy for children with persistent minimal residual disease. Citation Format: Gregor A. Rodriguez, Claudia P. Zapata, Anthony Sanchez, Nicolas A. De Cordoba, Beatriz E. Hawkins, Nadia G. Myrthil, Sarah A. Samuels, Amelia Bahamonde, Steven Vanni, Regina M. Graham. Withaferin A promotes ROS-mediated differentiation of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B41.

Systematic characterization of lncRNAs' cell-to-cell expression heterogeneity in glioblastoma cells.

Glioblastoma (GBM) is the most common malignant adult brain tumor generally associated with high level of cellular heterogeneity and a dismal prognosis. Long noncoding RNAs (lncRNAs) are emerging as novel mediators of tumorigenesis. Recently developed single-cell RNA-seq provides an unprecedented way for analysis of the cell-to-cell variability in lncRNA expression profiles. Here we comprehensively examined the expression patterns of 2,003 lncRNAs in 380 cells from five primary GBMs and two glioblastoma stem-like cell (GSC) lines. Employing the self-organizing maps, we displayed the landscape of the lncRNA expression dynamics for individual cells. Further analyses revealed heterogeneous nature of lncRNA in abundance and splicing patterns. Moreover, lncRNA expression variation is also ubiquitously present in the established GSC lines composed of seemingly identical cells. Through comparative analysis of GSC and corresponding differentiated cell cultures, we defined a stemness signature by the set of 31 differentially expressed lncRNAs, which can disclose stemness gradients in five tumors. Additionally, based on known classifier lncRNAs for molecular subtypes, each tumor was found to comprise individual cells representing four subtypes. Our systematic characterization of lncRNA expression heterogeneity lays the foundation for future efforts to further understand the function of lncRNA, develop valuable biomarkers, and enhance knowledge of GBM biology.

Syntheses and biological evaluation of 1,2,3-triazole and 1,3,4-oxadiazole derivatives of imatinib.

Three novel series of 1,2,3-triazole and 1,3,4-oxadiazole derivatives of imatinib were prepared and evaluated in vitro for their cytostatic effects against a human chronic myeloid leukemia (K562), acute myeloid leukemia (HL60), and human leukemia stem-like cell line (KG1a). The structure-activity relationship was analyzed by determining the inhibitory rate of each imatinib analog. Benzene and piperazine rings were necessary groups in these compounds for maintaining inhibitory activities against the K562 and HL60 cell lines. Introducing a trifluoromethyl group significantly enhanced the potency of the compounds against these two cell lines. Surprisingly, some compounds showed significant inhibitory activities against KG1a cells without inhibiting common leukemia cell lines (K562 and HL60). These findings suggest that these compounds are able to inhibit leukemia stem-like cells.

ATPS-47ORAL ADMINISTRATION OF THE AXL TYROSINE KINASE INHIBITOR BGB324 PROLONGS SURVIVAL OF GLIOBLASTOMA-BEARING MICE

Overexpression of the Axl receptor tyrosine kinase is associated with a negative outcome for glioblastoma patients. The aim of the study was to evaluate the effect of the specific Axl-inhibitor BGB 324 on glioma cell proliferation and migration in vitro as well as on orthotopic tumor growth in vivo. Axl expression was analyzed in adherent glioblastoma cell lines (N = 32) as well as in spherical stem-like glioblastoma cell lines (N = 9) by microarray and qPCR analyses, Western blot and flow cytometry. Axl was strongly expressed in glioblastoma cells with a proliferative, differentiated phenotype, such as G55 and U87, while its expression was weak in stem-like GBM cells. Full length Axl protein was present at the cell surface, while its soluble form sAxl was detectable in conditioned media and was released from the cells in a metalloproteinase-dependent fashion. The Axl ligand Gas6 was also present in conditioned medium, indicating an autocrine stimulation loop. In addition, unprocessed full length Axl was found in conditioned medium, suggesting an association of Axl with glioblastoma-derived exosomes. Erk1/2 and Akt phosphorylation induced by recombinant Gas6 was abrogated by interfering with Axl tyrosine kinase function using BGB324, resulting in a dose-dependent decrease in proliferation and cell migration. Most importantly, oral administration of 25 mg/kg BGB324 to tumor-bearing mice twice daily significantly increased the median survival of treated animals compared to vehicle controls for both G55 (23 vs 19 days, p < 0.0001) and U87 (26.5 vs 24days, p < 0.001) xenograft models. In summary, targeting Axl by oral administration of BGB324 appears a promising strategy for Axl-overexpressing glioblastoma cells that have undergone proliferative differentiation. The impact of Axl inhibition glioblastoma stem-like cells remains to be elucidated.

ATPS-48A HIGH-THROUGHPUT SCREEN IDENTIFIES microRNA-1300 AS A POTENTIAL THERAPEUTIC microRNA CAUSING CYTOKINESIS FAILURE AND APOPTOSIS IN GLIOBLASTOMA CELLS

INTRODUCTION: There is a pressing need for new therapeutic approaches for the treatment of glioblastoma (GBM). MicroRNAs are single-stranded 22-24nt non-coding RNAs which function by reducing translation or causing degradation of target mRNAs. MiR-1300 was identified in a high-throughput miRNA mimic library screen as a promising therapeutic candidate, inducing cell death. METHOD: To validate the cytotoxic effect of miR-1300, a panel of four established cell lines (U251,LN229,U373,KNS42)and two patient-derived stem-like cell lines were utilised for RTqPCR, imaging and flow-cytometry to assess endogenous expression levels, cellular morphology, apoptosis, and cell cycle delay respectively. Bioformatic approaches identified potential miR-1300 targets as mediators of this effect and validation of this involvement in the cytotoxic phenotype using siRNA knock-down is being investigated. Validation of the direct targeting by miR-1300 is being performed using 3' UTR Luciferase assays. RESULTS: Expression, of miR-1300, by transfection of oligonucleotide mimics, in our panel of GBM cells consistently led to G2M arrest followed by apoptosis within 72h. The associated phenotype is binucleation, indicative of cytokinesis failure.Preliminary data suggest this may be mediated through ECT2, a predicted target of miR-1300 known to be involved in formation of the cleavage furrow. CONCLUSION: We have identified a highly potent pro-apoptotic microRNA that may be exploitable for GBM therapy. Importantly, there is no detectable endogenous miR-1300 expression in any GBM samples or cell lines. On-going work will confirm the targets of miR-1300 that drive this effect as well as exploring potential approaches to deliver this as a biotherapeutic agent.

IMCT-12A PHASE 1 TRIAL OF VACCINATION WITH AUTOLOGOUS DENDRITIC CELLS PULSED WITH LYSATE DERIVED FROM AN ALLOGENEIC GLIOBLASTOMA STEM-LIKE CELL LINE FOR PATIENTS WITH NEWLY DIAGNOSED OR RECURRENT GLIOBLASTOMA

BACKGROUND: Dendritic cell vaccines are a promising treatment for glioblastoma. The glioblastoma stem-like cell subpopulation is refractory to standard chemotherapy and radiation treatment, but may be susceptible to immunotherapeutic targeting. METHODS: This single-institution phase I trial is designed to assess the safety, tolerability, and potential efficacy of an autologous dendritic cell vaccine pulsed with lysate derived from a glioblastoma stem-like cell line. Patients with newly diagnosed glioblastoma (Cohort A) receive standard radiation and temozolomide in addition to vaccine. Patients with up to third glioblastoma recurrence (Cohort B) receive vaccine alone, although patients previously treated with bevacizumab are allowed to continue bevacizumab on study. Prior to enrollment, patients must undergo a qualifying gross total or near-gross total resection. Treatment consists of a Vaccine Induction Phase (weekly vaccines x 4 – for Cohort A, given upon completion of radiation), followed by a Vaccine Maintenance Phase (vaccine every 8 weeks until supply depleted). Imaging is performed during Week 4 of the Induction phase and every 8 weeks thereafter until progression. The primary objective of safety and tolerability includes adverse event grading per NCI CTC. Clinical assessment includes progression-free survival at 6 months and overall survival. Immunological testing to assess cytotoxic T-lymphocyte response will be performed at predetermined intervals. Goal enrollment is 20 patients in each cohort. Stopping rules for safety and efficacy using Bayesian sequential design will be followed. RESULTS: To date, 18 patients (7 new GBM, 11 recurrent GBM) have enrolled in this trial. No adverse events related to vaccine have been noted. CONCLUSION: This single-institution phase I dendritic cell vaccine trial builds upon our previous research efforts by specifically targeting the glioblastoma stem-like cell subpopulation that is resistant to standard radiation and chemotherapy. Accrual is proceeding rapidly, and results will be published when available.

Abstract 706: Inflammatory and stem-like colorectal cell lines show differential response to MEK-162 and neratinib in combination

Abstract Background: Improvement of colorectal cancer (CRC) treatment depends on finding new effective agents and associated biomarkers that can predict patient response to drugs. Recent cell culture experiments show that a combination of MEK and pan ERBB inhibitors may be efficacious in treating CRC (Sun et al 2014). The purpose of this study was to determine whether a cell line's response to targeted agents would correlate with its mutation profile and/or gene expression subtype. Methods: K-RAS mutation status of 7 cell lines were identified and classified into stem-like or inflammatory subtypes using gene expression data from the cancer cell line encyclopedia and an algorithm described by Sadanandam et al. We tested 5 stem-like cell lines (SW480, SW620, HCT116, C2BBE1, HS675T) and 2 inflammatory cell lines (NCI-H747, SW837) for response to inhibitors of the RAS/RAF and PI3K pathways. MEK-162 and neratinib were tested as single agents and in combination, using cell viability and clonogenic assays. Results: Cell lines varied in response to the drugs when used as single agents. All cell lines were resistant to the EGFR inhibitor (Gefitinib) at concentrations ≥5 μM. Two pan ERBB inhibitors (neratinib and afatinib) showed IC50 values at or &amp;gt;4 μM and 2 MEK inhibitors (Selumetinib and MEK162) had IC50 values at or &amp;gt;1μM. The inflammatory cell lines, SW837 and NCI-H747, had greater sensitivity to the MEK inhibitors (IC50 value 1μM) while the KRAS mutant stem-like cell lines were relatively more resistant (SW480, SW620) with IC50 values ≥4-5 μM. Stem-like cell lines with WT-KRAS (C2BBE1, HS675T) were even more resistant; IC50s were not achieved at concentrations of 5 μM. Similar results were obtained in clonogenic growth assay. The MEK and pan ERBB inhibitor combinations were more effective at decreasing cell viability and inducing apoptosis in inflammatory cell lines than in stem-like cell lines. This combination effectively inhibited 60-70% of cell viability in inflammatory cell lines at a dose of 0.5 μM and 0.062 μM for neratinib and MEK-162 respectively. The lethality of this combination was the same even if the doses were reversed (0.5 μM MEK-162 and 0.062 μM neratinib). In contrast, we observed that this combination was not nearly as effective against stem-like cell lines irrespective of KRAS mutation status. Profiling of the phosphorylation status of proteins in the RAF-MEK-ERK signaling pathway after exposure to the MEK-162 plus neratinib combination showed a substantial inhibition of ERK phosphorylation in inflammatory cell lines but not in 4 of the 5 stem-like cell lines. Conclusion: This study demonstrates that colon cancer cell lines classified as the inflammatory subtype are sensitive to the combination of MEK-162 plus neratinib at concentrations at which cell lines of the stem-like subtype are resistant. The PA DOH disclaims responsibility for analysis, interpretations, or conclusions. Citation Format: Rekha Pal, Nan Song, Ashok Srinivasan, Samuel A. Jacobs, Soonmyung Paik, Katherine L. Pogue-Geile. Inflammatory and stem-like colorectal cell lines show differential response to MEK-162 and neratinib in combination. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 706. doi:10.1158/1538-7445.AM2015-706

Abstract 1409: Whole-cell and local viscoelasticity of stem-like and non-stem-like breast cancer cells

Abstract It has been shown that the mechanical properties of pathological cells are different from healthy ones. Therefore, mechanical characterization of cells may be useful for investigating mechanisms and progression of diseases. In the case of cancer, the finding that cancerous cells are considerably more deformable than normal ones provides the motivation for investigating rheological differences between stem-like and non-stem-like cancer cells. In this study, the viscoelastic properties of six breast cancer cell lines, three of which have been regarded (based on surface markers) as stem-like and three considered less stem-like, were measured using micropipette aspiration and particle tracking microrheology. In micropipette experiments, cancer cells were aspirated one-by-one at controlled pressures into small glass micropipettes and the length of the aspirated section of the cells was tracked as a function of time. The applied pressure was either increased linearly or increased suddenly to different levels and then held constant. The homogeneous elastic half-space model was used to interpret the viscoelastic properties of the different cell lines. The effect of pressure loading rate on cell viscoelasticity was examined by applying three different aspiration pressure rates of 9, 6, and 3 Pa/s. The apparent Young's modulus was found to depend on pressure loading rate, which suggests a more complex rheology than a standard viscoelastic solid. Above a threshold force level, cells were also found to enter the micropipette continuously in a fashion that is analogous to white blood cells, i.e. as a liquid drop with constant cortical tension. A comparison between the Young's moduli showed that at least some of the stem-like breast cancer cell lines were less stiff than less stem-like cell lines and more likely to exhibit the liquid drop-like behavior. The reduction in stiffness and change in rheological regime might be the result of lower actin filament density which allowed the stem-like cancer cells to be more deformable while undergoing extravasation and intravasation during the metastatic process. Regional rheological differences were also found using particle tracking microrheology. Our hypothesis is that the differences in the rheological properties might help to distinguish between stem-like cancer cells and non-stem-like ones, which could eventually lead to a new detection and diagnostic method in cancer research. Citation Format: Amina Alipour, Monica M. Burdick, David F.J. Tees. Whole-cell and local viscoelasticity of stem-like and non-stem-like breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1409. doi:10.1158/1538-7445.AM2015-1409

Abstract 399: Nanoparticle silencing of β1 and β3 integrins augments Dasatinib sensitivity

Abstract Triple negative breast cancers (TNBCs) are an aggressive subcategory of breast cancers that currently lack FDA-approved targeted therapies. Previous studies have i) further subcategorized TNBCs into 5 distinct subtypes, each with a preferential response to specific chemotherapies and ii) identified beta1 and beta3 integrins to be key mediators of breast cancer EMT and metastasis. We surveyed beta1 and beta3 integrin expression in the 5 subtypes of TNBC with and without treatment of their proposed chemotherapies by western blot analysis. Using nanoparticles, we delivered beta1 and beta3 integrin-specific siRNAs to silence the expression of both integrins either with or without treatment of the proposed chemotherapies, and evaluated the effect of these treatment regimens on EMT, invasion, and 3D outgrowth. We demonstrated that beta1 and beta3 integrins are robustly expressed in the Mesenchymal Stem-Like, Mesenchymal, and Basal-Like2 subtypes of TNBCs. Furthermore, we observed elevated expression of beta1 integrin upon Dasatinib treatment of the Mesenchymal Stem-Like subtype. Lastly, we determined that delivery of beta1 and beta3 integrin siRNAs sensitized breast cancer cells to Dasatinib treatment. Collectively, our data suggests that delivery of beta1 and beta3 integrin-specific siRNAs in combination with Dasatinib treatment efficiently eliminates the aggressive characteristics of Mesenchymal Stem-Like breast cancer cell lines. Citation Format: Jenny G. Parvani, Vinson Chu, Sarah Roelle, Zheng-Rong Lu, William Schiemann. Nanoparticle silencing of β1 and β3 integrins augments Dasatinib sensitivity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 399. doi:10.1158/1538-7445.AM2015-399

Role of PI3K/AKT pathways in mitomycin-mediated apoptosis of WB-F344 cells

To investigate the role of p38MAPK and PI3K/AKT pathways in mitomycin (MMC)-induced apoptosis in the liver stem-like cell line WB-F344. WB-F344 cells were exposed to MMC and apoptosis was evaluated by flow cytometry and DNA fragmentation. Phospho-MAPK and phospho-PI3K/AKT were detected by western blotting. MMC induced apoptosis in WB-F344 cells at 6h after addition of MMC; the maximum level of apoptosis was reached at 24h after MMC exposure. The apoptosis effects of MMC were concentration dependent and inhibited when the PI3K pathway was abolished by the specific inhibitor LY294002, but not inhibited when the p38MAPK pathway was abolished by inhibitor SB203508. Apoptosis of WB-F344 cells can be induced by MMC.Although MMC can activate both the PI3K/AKT and p38MAPK pathways, the apoptosis effect of MMC occurs via a PI3K pathway and is not dependent on the p38MAPK pathway.

Quantitative Relationships Between the Cytotoxicity of Flavonoids on the Human Breast Cancer Stem-Like Cells MCF7-SC and Their Structural Properties.

As some breast cancer-related deaths can be attributed to the metastasis of cancer stem cells, chemotherapeutic agents targeting breast cancer stem cells are of interest as a potential treatment. Flavonoids that exhibit cytotoxicity on breast cancer stem cells have rarely been observed. Thus, the objective of this study was to measure potential cytotoxic effects of 42 different flavonoids on the human breast cancer stem-like cell line, MCF7-SC. The relationship between flavonoid structural properties and cytotoxicity has not been reported previously; therefore, we determined quantitative structure-activity relationships using both comparative molecular field analysis and comparative molecular similarity analysis. Further biological experiments including Western blot analysis, flow cytometry, and immunofluorescence microscopy were also conducted on the most cytotoxic 8-chloroflavanone.

Secondary Data Analytics of Aquaporin Expression Levels in Glioblastoma Stem-Like Cells.

Glioblastoma is the most common brain tumor in adults in which recurrence has been attributed to the presence of cancer stem cells in a hypoxic microenvironment. On the basis of tumor formation in vivo and growth type in vitro, two published microarray gene expression profiling studies grouped nine glioblastoma stem-like (GS) cell lines into one of two groups: full (GSf) or restricted (GSr) stem-like phenotypes. Aquaporin-1 (AQP1) and aquaporin-4 (AQP4) are water transport proteins that are highly expressed in primary glial-derived tumors. However, the expression levels of AQP1 and AQP4 have not been previously described in a panel of 92 glioma samples. Therefore, we designed secondary data analytics methods to determine the expression levels of AQP1 and AQP4 in GS cell lines and glioblastoma neurospheres. Our investigation also included a total of 2,566 expression levels from 28 Affymetrix microarray probe sets encoding 13 human aquaporins (AQP0–AQP12); CXCR4 (the receptor for stromal cell derived factor-1 [SDF-1], a potential glioma stem cell therapeutic target]); and PROM1 (gene encoding CD133, the widely used glioma stem cell marker). Interactive visual representation designs for integrating phenotypic features and expression levels revealed that inverse expression levels of AQP1 and AQP4 correlate with distinct phenotypes in a set of cell lines grouped into full and restricted stem-like phenotypes. Discriminant function analysis further revealed that AQP1 and AQP4 expression are better predictors for tumor formation and growth types in glioblastoma stem-like cells than are CXCR4 and PROM1. Future investigations are needed to characterize the molecular mechanisms for inverse expression levels of AQP1 and AQP4 in the glioblastoma stem-like neurospheres.

TRIM28 and β-actin identified via nanobody-based reverse proteomics approach as possible human glioblastoma biomarkers.

Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.

NT-33 * NAD+ SYNTHESIS INHIBITION AS A THERAPEUTIC STRATEGY FOR GLIOBLASTOMA WITH MYC AMPLIFICATION

BACKGROUND AND OBJECTIVE: Oncoprotein Myc is a master transcription factor and plays a crucial role in human cancer pathogenesis and the stem-like phenotype of glioblastoma (GBM). Since dyregulated Myc in cancer drives altered glucose and glutamine metabolism promoting tumor growth, pathways involved in Myc-induced aberrant metabolism could offer therapeutic targets. The coenzyme Nicotinamide Adenine Dinucleotide (NAD+) is essential in redox reactions including glycolysis and the citric acid cycle. We sought to determine whether MYC-amplified GBM, as compared to MYC-non-amplified GBM, utilizes distinct NAD+ biosynthesis pathways and exhibits differential dependence on metabolic pathways that require NAD+. MATERIALS AND METHODS: A total of 11 patient-derived GBM stem-like cell (GSCs) lines established from surgical specimens were used. FISH analysis was performed to determine copy number status of MYC and MYCN. Expression of enzymes involved in NAD+ synthesis [e.g., nicotinamide phosphoribosyltransferase (NAMPT) and nicotinic acid phosphoribosyltransferase (NAPRT-1)] was tested with Western blot. NAD+ levels were measured after treating GSCs with FK866, a potent and specific small molecule inhibitor for NAMPT. The effects of pharmacological inhibition of NAMPT on GSC viability were determined by CellTiter Glo cell viability assay. RESULTS: Five (45.5%) GSCs had MYC (1) or MYCN (4) amplification. FK866 depleted NAD+ levels in GSCs. Treatment with FK866 potently inhibited in vitro growth of all the GSCs that harbored MYC/MYCN amplification with IC50 values in a low-nanomolar range (0.7 - 4.6 nM). In contrast, non-amplified GSCs as well as human normal astrocytes showed resistance to FK866. The MYC/MYCN amplified GSCs expressed low or undetectable levels of NAPRT-1, which is involved in the NAD+ synthesis pathway utilizing nicotinic acid. CONCLUSIONS: MYC/MYCN-amplified GSCs, but not non-amplified GSCs, were exquisitely sensitive to NAMPT inhibition. NAMPT may represent a promising therapeutic target for GBM with MYC amplification.