bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract A196: Characterization of stability and biological activity of the cancer gene therapy biologic SNS01 following storage at ambient and freezing temperatures
Authors: Taylor Catherine , Ye Bin , Liu Zhongda , Sun Zhong , Dondero Richard , Galton Bruce , Lust John , Donovan Kathleen , Thompson John 
Published: June 2012

Abstract
Introduction: The eukaryotic translation initiation factor 5A (eIF5A) is the only known protein to be regulated by post-translational addition of a hypusine residue. Both hypusinated eIF5A and the enzyme that mediates eIF5A hypusination have been identified as markers of neoplastic growth and metastasis. However, recent studies have indicated that, in its unhypusinated form, eIF5A is pro-apoptotic and thus functionally distinct from hypusine-modified eIF5A. In vitro cell studies and in vivo xenograft studies have demonstrated that simultaneous suppression of eIF5A expression and overexpression of a non-hypusinable mutant of eIF5A potently induces apoptosis in multiple cancer cell types. SNS01 is a nanoparticle specifically designed for the treatment of multiple myeloma. SNS01 is comprised of three components: a DNA vector containing a B-cell-specific (B29) promoter driving expression of a pro-apoptotic mutant of eIF5A (eIF5AK50R) that cannot be hypusinated; an siRNA that targets the native hypusinated eIF5A that promotes growth of cancer cells; and a synthetic polymer called polyethylenimine (PEI).
Methods: Ethidium-bromide agarose gel electrophoresis was used to detect heparin-mediated release of nucleic acids from nanoparticles. Dynamic light scattering (DLS) was used to assess the short-term stability of SNS01 by monitoring changes in size distribution, polydispersity, and zeta potential. In vitro biological activity of SNS01 was assessed using RT-qPCR to measure eIF5A and eIF5AK50R transgene expression as well as FACS detection of AnnexinV-FITC labeled apoptotic cells. Anti-tumoral activity of SNS01 was evaluated using a subcutaneous human myeloma model (KAS-6/1 cells) in SCID mice with twice-weekly intra-venous injections of SNS01.
Results: SNS01 nanoparticles were found to be relatively stable for as long as 48 hours at room temperature with no significant loss in nucleic acid integrity. The nanoparticles had an average diameter of 146.2 nm, a polydispersity of 0.287 and an average zeta potential of 35.3+0.7 mV. These parameters were quite stable over 48 hours. SNS01 could also be frozen at − 80°C for at least one month with no observable loss in biological activity. Twice-weekly intra-venous injections into myeloma tumor-bearing mice of a frozen SNS01 preparation was found to have anti-tumoral activity comparable to freshly prepared SNS01 (frozen SNS01: 89 % tumor inhibition, p = 0.00158; SNS01: 84 % tumor inhibition, p = 0.00003).
Conclusions: SNS01 is relatively stable at room temperature and was also found to be stable and biologically active following freezing.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A196.
Abstract A199: Oncogenic and drug-sensitive ERBB2 mutations in glioblastoma
Authors: Greulich Heidi , Chheda Milan , Chen Tzu-Hsiu , Tanaka Kumiko , Dutt Amit , Wong Kwok-Kin , Hahn William , Meyerson Matthew 
Published: June 2012

Abstract
The Cancer Genome Atlas (TCGA) Consortium recently completed a multidimensional analysis of glioblastoma, characterizing somatic alterations across a large sample set, and identifying genetic lesions that likely contribute to tumor fitness (TCGA Network, Nature, 2008). However, functional experiments are required to determine whether significantly altered genes are in fact oncogenes or tumor suppressor genes in glioblastoma. We experimentally analyzed mutations in one of the significantly mutated genes, ERBB2. Unlike ERBB2 mutations in lung adenocarcinoma, ERBB2 mutations in glioblastoma are primarily located in the extracellular region, clustering around the domain that mediates receptor dimerization. We have determined that a subset of the reported mutations are gain-of-function and oncogenic, require kinase activity for oncogenic transformation, and are sensitive to specific small molecule kinase inhibitors. This discovery has therapeutic implications for glioblastoma patients who harbor somatic ERBB2 mutations.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A199.
Abstract A202: RNAi as a tool to identify novel molecular vulnerabilities in myeloid leukemias
Authors: Tibes Raoul , Bogenberger James , Hagelstrom Tanner , Buechel Megan , Azorsa David O. 
Published: June 2012

Abstract
Therapies and outcomes of patients with Acute Myeloid Leukemia (AML) and advanced myelodysplastic syndrome (MDS) remain very poor and AML is one of the deadliest cancers in relation to incidence. Mostly, because critical targets regulating myeloid cell viability have not been identified. Genomic profiling approaches, including gene expression or sequencing may identify genes and proteins involved in leukemogenesis. However candidates identified with these more “traditional” methods may not necessarily be good drug targets or represent molecular vulnerabilities that can be targeted. Therefore, we pursued a functional genomcis approach using RNAi to identify novel targets that can be exploited for drug development and discovery in order to improve treatment strategies in AML and MDS.
First, we established a platform for High-Throughput RNAi (siRNA) gene silencing in typical difficult to transfect suspension cells. Using reverse lipid based transfection conditions we were able to establish conditions that achieve up to 80–90% transfection as measured by a universal silencing control. After successful establishment of our siRNA platform in 3 cell lines, we performed several siRNA screens of the human kinome and other cancer associated genes (i.e. apoptotic and cell cycle genes) with siRNA's alone or in combination with 5-Azacytidine or Cytarabine. In this abstract we will present our experience with siRNA screens in myeloid cells as well as data to support the tremendous opportunity the RNAi approach holds in identifying direct druggable targets in acute leukemias. Our ultimate goal is to develop a “Functional Genomics Pathway Vulnerability Profiling” in AML. Functional genomics using RNAi provide a fast and attractive approach to identify molecular targets in AML/MDS and this approach holds particular promise to develop and design rational (combination) therapies that can be rapidly translated into the clinic.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A202.
Abstract A204: Investigating a novel antitumor molecular target using a chemical proteomic strategy
Authors: Evans Hayley R. , Rong Dawen , Phillips Roger M. , Sutton Chris W. , Wheelhouse Richard T. 
Published: June 2012

Abstract
Three novel, synthetic biarylheterocycles bearing imidazole terminal groups were discovered with extreme potency (IC50 16–640 nM) against the drug-resistant (Mitomycin C and EO9) colon carcinoma cell line BE, and also the human ovarian carcinoma A2780 and human non-small cell lung cancer H460 cell lines. Notably, this biological activity was independent of duplex DNA binding affinity. The compounds were tested in the NCI 60-cell line panel and COMPARE analysis suggests they are targeting the product of a ‘gene-like sequence’ of unidentified function.
The identity of the target proteins was explored using an affinity chromatography proteomic strategy. Bespoke affinity matrices were prepared in which test compounds were attached to a solid support through a biotin tag. A synthetic route to compounds containing a biotin moiety in place of one of the imidazole sidechains was developed. Chemosensitivity studies confirmed that the biotinylated compounds retain their activity (IC50 6.25 µM in a susceptible cell line, compared with &gt; 100 µM for an insensitive cell line).
The biotinylated ligands were exposed to protein extracts from the susceptible cell lines and ligand-protein complexes collected using a streptavidin-activated affinity column. Bound proteins were eluted from the column and separated using SDS-PAGE. Proteins were characterised by MALDI MS/MS and identified using database searches.
In parallel, the mechanism of action of the compound series has been investigated using NCI 3D MIND data mining. This suggests the mechanisms of action to be associated with phosphatase- and kinase-mediated cell cycle regulation.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A204.
Abstract A207: Whole transcriptome profiling in upper aerodigestive tract cancers
Authors: Dinali Mohamed El , Braegelmann Johannes , Stricker Thomas , Brown Christopher , Salgia Ravi , Vokes Everett , White Kevin , Seiwert Tanguy 
Published: June 2012

Abstract
Background: RNA-seq is a powerful technology that allows to obtain sequence and expression information simultaneously. We applied this technology to 4 upper aerodigestive tract cancers.
Methods: RNA from four upper aerodigestive tract tumor specimens was extracted and sequencing libraries constructed. Samples were analyzed using an Illumina Genome Analyzer with a paired end module (36 or 54 base read length). Raw data was processed with a proprietary data pipeline. Potential mutations were filtered using both in house and external bioinformatic solutions, involving previously established databases of mutations (COSMIC) and SNPs (dbSNP), and removal of ancestral alleles identified from multiple sequence alignments. These SNPs were then parsed via in house scripts to determine whether the SNPs were present in coding regions, 3′UTR, 5′UTR, or in splice acceptor/donor sites. The coding SNPs were further parsed to determine which SNPs result in non-synonymous changes. RNA-Seq expression data was analyzed using Partek Genomics Suite and compared to microarray based expression data. Pathway analysis was performed using GeneGO Metacore and DAVID.
Results: 500Mb to 2GB of data were obtained per sample. Between 700 and 2000 nsSNPs were identified, as well as a large number of alteration in the 3′ and 5′ untranslated regions. Genetic alterations in several commonly mutated genes were identified including TP53, ErbB2, H-RAS, and Cyclin D1. SNPs were enriched in pathways commonly involved in upper aerodigestive tract tumors including cell cycle control, cytoskeleton, and receptor tyrosine kinases. Furthermore we screened for viral sequences identifying one sample that was HPV16 positive. RNA-seq based and microarray based expression data generally correlated, but significant differences were also observed, that were at least partly due to the limited number of RNA-seq reads.
Conclusion: Cancer transcriptome sequencing is a promising and cost effective approach for identifying mutations and obtaining expression analysis simultaneously. Transcriptome sequencing holds promise as a readily available platform for assessing potential treatment targets in a specific tumor.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A207.
Abstract A209: Comprehensive analysis of the MET protooncogene promoter regulation
Authors: Trzyna Elzbieta , Duleba Marcin , Faryna Marta , Majka Marcin 
Published: June 2012

Abstract
MET protein, known as hepatocyte growth factor (HGF) receptor, is a member of receptor tyrosine kinase family. It exerts its action through various signal transduction pathways leading to cell proliferation, scattering and acquisition of migratory phenotype. The MET mutations or protein overexpression are believed to play a crucial role in cancer growth and metastasis formation, acting by upregulating proliferation, inhibiting apoptosis and enhancing migrative capacity of the cells. The expression of MET is driven by several regulatory elements. Its promoter is characterized by an extremely high GC content and lack of TATA box sequence. The current knowledge of the mechanism of transcriptional control exerted on the MET promoter and the putative mutations leading to an increased protein expression is very limited.
Therefore, the aim of our project was to investigate and characterize the promoter region.
The first stage of the investigation involved establishing and comparing the promoter sequence from several multiple myeloma cell lines, as well as from CD138+ cells collected from multiple myeloma patients and from healthy individuals. The ulterior phases of the project included examination of the CpG islands methylation pattern in multiple myeloma cell lines. Results were compared to the expression of MET gene in cell lines and healthy individuals. The third aim of our project is to identify and characterize protein factors binding to the promoter sequence in multiple myeloma cell lines.
Experiments were performed by the mean of DNA sequencing of DNA cloned to vector pCR2.1. Methylation analysis required genomic DNA treatment with Sodium Bisulphite and cloned into the pCR2.1 vector and sequenced. “Protein fishing” was performed by the use of cell lysates incubated with biotinylated MET promoter specific probe and streptavidin magnetic beads and separated on magnetic µ-columns. Proteins were analyzed in one- and twodimensional electrophoresis and silver stained.
Promoter sequencing revealed some dispersed and random alterations in myeloma and healthy individuals samples. Observed the most common substitutions −304C&gt;A and +206C&gt;G with higher occurrence in cancer cell lines. Methylation analysis of multiple myeloma cell lines showed lower methylation of MET promoter sequence than control DNA.
“Protein fishing” analysis resulted in differences in bands profile between cytoplasmic and nuclear samples of different cell lines. The dots visualised in 2-DE electrophoresis, possible transcription factors, are currently under investigation.
The identified mutations, methylation analysis and proteins profile observed so far are to be further investigated to assess their relevance for the MET transcriptional regulation.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A209.
Abstract A211: Fragment-based drug discovery of the synthetic small molecule HSP90 inhibitor AT13387
Authors: Murray Christopher W. , Carr Maria G. , Chessari Gianni , Congreve Miles , Coyle Joseph E. , Day Philip J. , Fazal Lynsey , Frederickson Martyn , Graham Brent , Lewis Jonathan , McMenamin Rachel , O'Brien Alistair , Patel Sahil , Williams Glyn , Woodhead Andrew J. , Woolford Alison J. 
Published: June 2012

Abstract
Heat Shock Protein 90 (HSP90) is a member of a family of molecular chaperone proteins which directs the folding of polypeptides into functional configurations affecting stabilisation and activation. Many of these proteins are oncogenes regulating tumor cell growth, survival and apoptosis. This poster will focus on the screening and medicinal chemistry work that led to the identification of AT13387, a high affinity HSP90 inhibitor that is currently in clinical trials for the treatment of cancer.
A fragment screening campaign was conducted against the N-terminal domain of HSP90 to detect very low molecular weight compounds (Molecular Weight &lt;250 Da) that bound to the ATPase active site. The screening produced a small fragment which was co-crystallised with HSP90 and had an affinity of 790µM. Three iterations of structure-guided medicinal chemistry led to the identification of a lead compound with 0.5nM affinity for the enzyme, good cell activity and confirmed mechanism of action in cells. The fragment to lead optimisation increased affinity by over a million fold but resulted in a molecule that was only 6 heavy atoms larger than the fragment starting point. Such an efficient optimisation campaign is unprecedented in the field of fragment-based drug discovery.
Subsequent lead optimisation focussed on the improvement of in vivo distribution properties via the addition of basic moieties to the lead molecule. These compounds showed encouraging in vivo pharmacology and biological profiles, and further medicinal chemistry work led to the discovery of AT13387, an inhibitor with sub-nanomolar affinity, prolonged duration of action and excellent in vivo anti-tumor efficacy.
This poster represents first disclosure of the structure of AT13387 and illustrates how a fragment-based drug discovery approach can be efficiently used to discover compounds suitable for clinical testing in oncology.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A211.
Abstract A212: A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor-induced apoptosis in HCT116 colon carcinoma cells
Authors: Massey Andrew J. , Borgognoni Jennifer , Browne Helen , Daniels Zoe , Dokurno Pawel , Drysdale Martin J. , Francis Geraint L. , Graham Christopher J. , Macias Alba T. , Matassova Natalia , Murray James B. , Parsons Rachel , Shaw Terry , Wang Yikang , Wood Mike , Williamson Douglas 
Published: June 2012

Abstract
The role of the 70 kDa heat shock protein isoforms (Hsc70 and Hsp70) in cancer development and progression through their ability to inhibit apoptosis and via their role as Hsp90 co-chaperones has been well documented. Dual targeting of Hsc70 and Hsp70 with siRNA has previously been demonstrated to induce proteasome-dependent degradation of Hsp90 client proteins and extensive tumor specific apoptosis as well as the potentiation of tumor cell apoptosis following pharmacological Hsp90 inhibition. The design and synthesis of novel adenosine-derived inhibitors of Hsp70, guided by modelling and X-ray crystallographic structures of these compounds in complex with Hsc70/BAG-1, has been described.1 These were the first inhibitors described to target the ATPase binding domain of this family of chaperones. Many of these compounds exhibited submicromolar affinity for Hsp70, were highly selective over Hsp90, and displayed in vitro activity against a variety of human tumor cell lines. We further describe the in vitro mode of action of one of the most potent analogues, VER-155008 in HCT116, HT29, BT474 and MDA-MB-468 carcinoma cell lines. Cell proliferation, cell cycle, cell apoptosis and caspase 3/7 activity was determined for VER-155008 in the absence or presence of small molecule Hsp90 inhibitors. VER-155008 inhibited the proliferation of human breast and colon cancer cell lines with GI50s in the range 5.3 to 14.4 M, and induced Hsp90 client protein degradation in both HCT116 and BT474 cells. As a single agent, VER-155008 induced caspase-3/7 dependent apoptosis in BT474 cells and non-caspase dependent cell death in HCT116 and HT29 cells. VER-155008 potentiated the apoptotic potential of the small molecule Hsp90 inhibitors VER-821602 and 17-AAG in HCT116 but not HT29 or MDA-MB-468 cells. In vivo, VER-155008 demonstrated rapid metabolism and clearance, along with tumor levels below the predicted pharmacologically active level. These data suggest that as a single agent, small molecule inhibitors of Hsc70/Hsp70 phenotypically mimic the cellular mode of action of a small molecule Hsp90 inhibitor and can potentiate the apoptotic potential of a small molecule Hsp90 inhibitor in certain cell lines. The factors determining whether or not cells apoptose in response to Hsp90 inhibition or the combination of Hsp90 plus Hsc70/Hsp70 inhibition remain to be determined.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A212.
Abstract A224: Noninvasive PD markers of a pyruvate dehydrogenase kinase inhibitor, dichloroacetate, in human colon carcinoma xenografts
Authors: Chung Yuen-Li , Troy Helen , Payne Geoffrey S. , Stubbs Marion , Judson Ian R. , Griffiths John R. , Leach Martin O. 
Published: June 2012

Abstract
Pyruvate dehydrogenase (PDH) is the key mitochondrial enzyme that determines whether pyruvate formed during glycolysis from glucose will be metabolised to lactate or oxidised in the TCA cycle. Its regulator, pyruvate dehydrogenase kinase (PDK) negatively regulates PDH by phosphorylation. Dichloroacetate (DCA) is an inhibitor of PDK and treatment with DCA decreased proliferation and growth in cancer cells and solid tumors (1). The aim of this work was to develop a non-invasive and robust pharmacodynamic (PD) biomarker for tumor response following PDK inhibition.
Human HT29 (colon) carcinoma xenografts were examined using in vivo1H- and 31P- magnetic resonance spectroscopy (MRS), before and after 3 days of DCA treatment (200mg/kg via po). Controls were treated with vehicle alone (water). Metabolic profiles of tumor extracts were measured by high resolution in vitro 1H- and 31P-MRS.
Significant tumor growth inhibition (p&lt;0.001) was observed in HT29 xenografts following 3 days of DCA treatment when compared with vehicle-treated controls. In vivo, significant decreases in ratios of phosphomonoester/total phosphorus signal (P=0.01) and total choline/water signal (P=0.05) were found in DCA-treated HT29 xenografts. The in vivo results were confirmed by significantly lower phosphocholine (PC) (P=0.02), glycerophosphocholine (P=0.01) and glycerophosphoethanolamine (P=0.05) levels, as found by 31P-MRS of tumor extracts from DCA treated animals when compared with controls. Increases in leucine (P=0.04), iso-leucine (P=0.02), valine (P=0.04) and succinate levels (P=0.03) and lower ATP (P=0.003), glucose (P=0.03) and creatine (P=0.05) levels were also found in DCA-treated HT29 tumor extracts when compared with controls.
The drop in PC and other choline-related metabolites following DCA treatment is probably due to tumor stasis caused by the PDK inhibition, since DCA upregulates the mitochondrial membrane potential, induces apoptosis, decreases proliferation and inhibits tumor growth (1). These and the non-phospholipid changes in the metabolic profiles including lower ATP are consistent with previous findings where similar metabolic profiles were associated with antiangiogenic effects induced by LAQ824 (2).
The phospholipid changes may have potential as surrogate non-invasive markers for determining tumor response following treatment with DCA or other PDK inhibitors.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A224.