bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A180: Tumor heterogeneity in glioblastoma is actively maintained by a paracrine circuit driven by mutant EGFR
Authors: Inda Maria-del-Mar , Bonavia Rudy , Sah Dinah W.Y. , Hadwiger Peter , Tan Pamela , Cavenee Webster K. , Furnari Frank
Published: June 2012

Glioblastoma is typified by a heterogeneous composition of diverse cytological subtypes carrying a variety of gene alterations. An example of heterogeneity that is present in >50% of cases is amplification of the epidermal growth factor receptor (EGFR) gene. This amplification is often accompanied by structural alterations leading to expression of a variant form of the gene, EGFR (also referred to as EGFRvIII, deltaEGFR, EGFR-de2-7 and EGFR*), which conveys enhanced tumor aggressiveness. This potent tumor-promoting function of EGFR would suggest that it should be the predominant amplified receptor in clinical samples, however, paradoxically, EGFR is usually present as minor and focal populations within tumors having more homogeneous wtEGFR amplification. This disconnect between tumorigenic potential and the frequencies and proportions of the amplified mutant EGFR and wtEGFR in GBM might arise if mutant EGFR occurs later in tumor progression where it not only enhances the tumorigenicity of cells which express it, but also potentiates, the proliferation of neighboring cells expressing amplified wtEGFR. If this were so, the potentiation loop might provide an attractive and novel therapeutic target. Here, we tested and validated the hypothesis that engraftment of a minor population of EGFR-expressing glioma cells can dramatically enhance the tumorigenicity of tumors cells expressing wtEGFR through paracrine stimulation. In support of this general notion, EGFR-driven pro-tumorigenic enhancement was lost upon siRNA-mediated knockdown of IL-6, and conversely, glioma cells engineered to over-express IL-6 enhanced the tumorigenicity of wtEGFR-expressing cells. These findings demonstrate that the molecular heterogeneity of GBM can be actively maintained, suggesting a coordinated strategy for therapeutic intervention.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A180.
Abstract A181: Dissecting the crosstalk between wt and mutant EGFR in glioblastoma multiforme
Authors: Bonavia Rudy , Inda Maria-del-Mar , Johns Terrance , Cavenee Webster , Furnari Frank
Published: June 2012

Glioblastoma Multiforme (GBM) exhibits intratumoral heterogeneity on the morphological and molecular levels, a hallmark feature which may contribute to its poor response to targeted therapy. A powerful oncogenic event in GBM is amplification and rearrangement of the epidermal growth factor receptor (EGFRvIII variant, deleted for exons 2–7, hereafter EGFR), yet notably, only a minority of primary tumor cells possess this signature lesion while the remainder maintain expression of the wildtype EGFR (wtEGFR). We observed that tumorigenicy of wtEGFR expressing cells can be significantly enhanced by the presence of EGFR-expressing cells within the tumor, suggesting that soluble factors produced by the latter cells might mediate this potent tumor enhancement. Here we show that in vitro treatment of U87 cells overexpressing wtEGFR (U87wt) with conditioned media from U87 cells overexpressing EGFR (U87) resulted in the activation of wtEGFR, STAT3, Akt, and Erk1/2. Surprisingly, U87 failed to demonstrate any increase in EGFR ligand production that could explain the enhanced wtEGFR phosphorylation. To identify the molecular mediators of this transactivation we analyzed the expression in U87 of cytokines of the IL-6 family, which are known potent activators of STAT3. We found a robust enhancement of the expression of IL-6 and LIF in U87 , compared to the parental cell line. Use of neutralizing antibodies allowed us to demonstrate that these candidate cytokines are responsible not only for the activation of STAT3, but also of the EGFR; results of which were confirmed by antibodies that block accessibility of their common gp130 receptor subunit. The analysis of other similar cellular systems suggested that, although not necessarily through the same cytokine(s), the more tumorigenic cells overexpressing EGFR generate signals to neighboring cells by engaging gp130, which is responsible for the activation of EGFR and STAT3. The importance of this circuitry in vivo was further demonstrated when, by knocking down gp130 expression in U87wt, we completely prevented the tumor growth enhancement usually observed in mixed composition tumors. These observations suggest gp130 as a potential new target for the treatment of gliomas.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A181.
Abstract A183: Ex vivo imaging of EGF receptor-mediated signal transduction for prediction of anti-EGFR treatment efficacy
Authors: Mishima Yuji , Matsusaka Satoshi , Terui Yasuhito , Mishima Yuko Matsumoto , Kuniyoshi Ryoko , Hatake Kiyohiko
Published: June 2012

Objectives: EGFR is a promising treatment target in solid tumors. Two major types of anti-EGFR agents have entered the clinical setting: antibodies and small-molecule EGFR TKIs. EGFR-TKI inhibits autophosphorylation of EGFR by binding to ATP binding site of tyrosine kinase domain of EGFR and interrupts the signal transduction. On the other hand, anti-EGER therapeutic antibodies interrupt EGFR signaling by blocking ligand binding. In addition, as for antibodies, the effects of the ADCC are also expected. In this study, we aimed at developing a method to estimate the effectiveness of anti-EGFR agents by analyzing these mechanisms using ex vivo imaging technique.

Methods: To establish a method that confirms the inhibition effect of the EGFR-signal transduction, we prepared fluorescent-labeled ligand and developed the imaging strategy to visualize membrane binding and subsequent internalization of labeled EGF. In addition, fluorescent-labeled NK-based effector cells that have been established by introducing fcgr3a gene into a NK leukemia cell line were used for characterization of ADCC susceptibility of individual tumor cells. In our strategy, a continuous confocal laser scanning microscopy was used for tracing fluorescent-labeled EGF or effector cells as well as tumor cell death.

Results: Fluorescent probe bearing EGF was highly specific and potent in the binding and activation of the EGF receptor, being rapidly internalized into endosomes. Pretreatment with therapeutic anti-EGFR antibody completely inhibited the ligand-binding and subsequent events. Meanwhile, EGFR TKIs partially prevented endocytotic step. K-ras mutation status did not significantly affect the ligand binding and internalization. The imaging analysis of the ADCC using primary colorectal cancer specimens revealed that cetuximab-coated tumor cells were gradually damaged by direct interaction of effector cells. ADCC susceptibility of individual tumor cells has varied to some extent.

Conclusion: We have established imaging methods of the early events of EGFR signaling pathway and ADCC reaction. This imaging method soffer the rapid reliable means that analyze the effect of the EGFR targeted agent to the individual tumor cells. By combining with already established prognostic factors such as K-ras mutation status or Fc receptor polymorphism, our imaging analysis will propose novel strategy to predict an individual treatment prognosis more precisely.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A183.
Abstract A185: Phase I clinical, pharmacokinetic, and pharmacodynamic study of SB939, an oral histone deacetylase inhibitor (HDACi), in patients with advanced solid tumors
Authors: Siu Lillian L. , Hotte Sebastien J. , Chen Eric X. , Hirte Hal W. , Powers Jean , Stayner Lee-Anne , Iacobucci Anne , Novotny-Diermayr Veronica , Zhu Joy , Eisenhauer Elizabeth A.
Published: June 2012

Background: SB939 is an orally available, potent, competitive HDACi selective for Class I, II and IV histone deacetylases. Preclinical evaluation of SB939 in a broad spectrum of xenograft mouse models revealed dose-dependent tumor growth inhibition. In the HCT116 colorectal cancer model, SB939 showed superior activity compared to other HDACi and prolonged histone H3 acetylation (acH3) in tumor tissues lasting ≥ 24 hours.

Methods: Patients (pts) with advanced solid malignancies were enrolled into 7 dose levels (DL). At DL1, SB939 was taken on days 1-3 and 15-17 every 4 weeks, then on days 1-5 and 15-19 for other DLs. Detailed PK sampling was performed on cycle 1 days 1 and 5 (for DL1 on days 1 and 3). Peripheral blood mononuclear cells (PBMC) were collected on cycle 1 at various time points for determination of acH3 levels using a validated Western blot assay. MTD was defined as dose with ≥ 2/3–6 pt with DLT.

Results: To date, 28 pts have received a total of 72 cycles: median age 62 (range 48–88); F:M = 10:18; ECOG 0:1:2 = 7;17:4; tumor types = colorectal (15), neuroendocrine (3), gastric (2), lung (2) and others (6); prior chemotherapy regimens 0:1:2:3+ = 5:1:6:16. The most frequent non-hematologic adverse events (AE) of at least possible attribution to SB939 were (% of pts with all grade/gr 3+): fatigue (46%/7%), nausea (29%/0%), anorexia (21%/4%), vomiting (18%/4%) and diarrhea (11%/0%). Hematologic AE were all mild to moderate. DLT occurrence is described below, with the 90 mg DL declared MTD. PK analysis showed dose-proportional increases in AUC and Cmax. Elimination half-life is 6–9 hours. PBMC analysis for acH3 (lysine9/14) showed highest levels at 3 hours post-dose with return to baseline by 24 hours. The average relative acH3 levels at 70 mg dose level in this study are comparable to those obtained at 60 mg dose level in another phase I study of SB939, which evaluates a different schedule of thrice weekly (every other day) for 3 out of 4 weeks. Four pts (14%) received 4 or more 4-week cycles of SB939 (range 6–10).

Conclusions: SB939 is well tolerated at up to 70 mg on this schedule of 5 consecutive days every 2 weeks. The 70 mg DL is being expanded and will likely be the RP2D. Phase II trials in prostate cancer and sarcoma are planned.

Dose level Schedule Total no of cycles No of pts with DLT/Total no of pts DLT Other significant toxicity 10 3 days 6 0/3 - - Q2W 10 5 days 16 1/6^ - - Q2W 20 5 days 18 1/6 Gr 3 - Q2W myositis 30 5 days 19 0/4 - - Q2W 50 5 days 6 0/4 - - Q2W 70 5 days 5 0/3 - - Q2W 90 5 days 2 1/2* Gr 3 2 gr 2 N/V Q2W fatigue 1 gr 2 QTc ^ gr 3 bilirubin rise subsequently declared not drug related * neither pt completed a full cycle

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A185.
Abstract A187: HDAC inhibitor SNDX-275 enhances herceptin-mediated therapeutic effects on HER2-overexpressing breast cancer cells
Authors: Huang Xiaoping , Wang Shuiliang , Lee Choon-Kee , Ordentlich Peter , Liu Bolin
Published: June 2012

Numerous studies indicate that deregulation of histone acetylation and deacetylation plays an important role in aberrant gene expression in human cancers. The Histone deacetylases (HDAC) have become attractive therapeutic targets, and many inhibitors of HDAC are actively under investigation as potential anti-cancer agents. SNDX-275 is an oral, class I HDAC inhibitor currently in multiple phase II clinical trials. We have recently reported that SNDX-275 selectively induces apoptosis of HER2-overexpressing breast cancer cells. Herceptin®, a humanized monoclonal antibody against HER2 receptor, has been successfully used in breast cancer patients with HER2-overexpressing tumors. In the current studies, we have investigated the potential synergism of SNDX-275 and Herceptin on inducing growth inhibition and/or apoptosis in HER2-overexpressing breast cancer cells, focusing on their combinational effects on the expression of both HER2 and HER3, and inactivation of downstream signaling pathways.

The cell proliferation (MTS) assays showed that SNDX-275 significantly enhanced Herceptin-induced growth inhibition in HER2-overexpressing breast cancer cells as compared to the treatment with either agent alone. While SNDX-275 (0.5 µM) alone clearly decreased the levels of HER3 with little effect on HER2 levels, Herceptin (20 µg/ml) alone mainly lowered the expression levels of HER2 in the HER2-overexpressing SKBR3, BT474, and MDA-MB-453 cells. The combinations of SNDX-275 (0.5 µM) and Herceptin (20 g/ml) resulted in a dramatic reduction of both HER2 and HER3 levels, and subsequently led to inactivation of HER2 tyrosine kinase evidenced by western blot analysis showing reduced levels of both P-HER2 and P-HER3. On the downstream signaling pathways, neither the combination nor either agent alone had inhibitory effects on MAPK. In contrast, the combinations of SNDX-275 and Herceptin significantly decreased the levels of P-Akt in SKBR3, BT474, and MDA-MB-453 cells as compared to either agent alone. It appeared SNDX-275 induced expression of p21waf1, and Herceptin upregulated p27kip1, however, their combinations had no further inductions on either of these two CDK inhibitors. Moreover, apoptotic-ELISA and western blot analysis on PARP cleavage revealed that the combinations of SNDX-275 and Herceptin promoted more cells undergoing apoptosis as compared to SNDX-275 alone.

Taken together, our results indicated that SNDX-275 and Herceptin synergistically induced growth inhibition and apoptosis in HER2-overexpressing breast cancer cells through combined downregulation of HER2/HER3 levels, inhibition of HER2 tyrosine kinase, and inactivation of the PI-3K/Akt signaling. These data suggest that further evaluation of SNDX-275 in HER2 positive breast cancer may be warranted.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A187.
Abstract A195: Inhibition of angiogenesis and growth of HER2-overexpressing tumors by SV40 T/t-common polypeptide
Authors: Hsueh Shu-Ping , Hsu Wei-Bin , Wang Won-Bo
Published: June 2012

HER2 overexpression in various tumors results in increased angiogenesis, metastasis and reduced survival. Previously we reported that simian virus 40 T/t-common polypeptide can repress HER2 expression and specifically induce apoptosis in HER2-overexpressing cancer cells. In this study, we further explored the effect of T/t-common on tumor angiogenesis. HER2-overexpressing cancer cells were either infected with T/t-common-carrying adenovirus (rAd-T/t) or vector virus (rAd-V). The supernatant of infected cells were then used in the Tanswell migration, tube formation, and Matrigel plug assays to evaluate its ability to induce angiogenesis. Our data indicate that conditioned medium from rAd-T/t-infected cells had much lower ability to induce the migration and tube formation of endothelial cells and to induce blood vessel formation in Matrigel plugs than that from rAd-V-infected cells. T/t-common was found to be able to inhibit the expression of pro-angiogenic factor VEGF-A through inhibiting the HER2-ERK-HIF-1 signaling pathway. Moreover, T/t-common could induce the expression of anti-angiogenic factor TSP-1 through activating the HER2-p38 pathway. Finally we show that infection of HER2-overexpressing SK-OV-3 xenograft tumors by rAd-T/t virus could lead to inhibition of tumor growth and microvessel formation in NOD/SCID mice. Together, these data suggest that T/t-common is a potential gene therapeutic agent that can inhibit the growth and angiogenesis of HER2-overexpressing tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A195.
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

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

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

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

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 > 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

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

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>A and +206C>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

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 <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

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

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<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.
Abstract A227: Positron emission tomographic (PET) imaging of activated matriptase, a marker for cancer progression
Authors: Choi Julia C. , Hausner Sven H. , Gagnon M. Karen J. , Kukis David L. , Holland Jason P. , Lewis Jason S. , Lin Chen-Yong , Johnson Michael D. , Sutcliffe Julie L.
Published: June 2012

The serine protease matriptase has been implicated in epithelial cancers, has been found in ∼45% of node-negative breast cancers at high levels, and is indicated as a biomarker for survival independent of HER-2/neu. While in vitro methods are invaluable, few breast cancer cell-lines express matriptase, though 83% of breast cancer patients are positive for matriptase—this suggests that imaging the in vivo behavior of matriptase may aid in understanding its role in the context of the tumor system. Specifically, activated matriptase is associated with cancer progression. We have developed radiotracers against activated matriptase for in vivo imaging using PET.

M69, an antibody against activated matriptase, was functionalized to capture [64Cu]copper or [89Zr]zirconium: 64Cu-TETA-M69 and 89Zr-DF-M69 were evaluated in a mouse model of human breast cancer. A dox-regulable pair of PyVmT cell-lines was bilaterally introduced into nude female mice to generate matriptase-positive and control tumors. Mice were fed dox chow ad libitum, administered radiotracer, imaged using microPET at 1–4d p.i.; and through 336h for 89Zr; corresponding biodistribution was performed.

PET images indicated specific tumor retention. Biodistribution at 4d revealed both 64Cu-TETA-M69 and 89Zr-DF-M69 retained two-fold uptake ratios for the positive tumor over control; this was also observed at 336h for 89Zr-DF-M69. Immunohistochemistry on FFPE tissues confirmed human matriptase expression in the positive tumor.

In summary, we have developed two novel radiotracers and performed the first in vivo imaging of activated matriptase. This approach has the potential for imaging metastasis, the primary cause of mortality in breast cancer patients.

Table 1 Tumor-to-liver ratios from biodistribution at 96h for activated matriptase. Radiotracer Matriptase-positive tumor Control tumor 64Cu-TETA-M69 2.55 ± 0.41 1.54 ± 0.33 89Zr-DF-M69 3.79 ± 0.25 1.88 ± 0.33

Table 2 Tumor-to-blood ratios from biodistribution at 96h for activated matriptase. Radiotracer Matriptase-positive tumor Control tumor 64Cu-TETA-M69 1.67 ± 0.39 1.01 ± 0.30 89Zr-DF-M69 1.97 ± 0.40 0.98 ± 0.45

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A227.
Abstract A233: Generation of in vitro and in vivo tumor models driven by insulin-like growth factor receptor (IGF1R) and their use in the development of OSI-906, a selective IGF1R inhibitor
Authors: Liu Qing , Lorena Lerner , Tao Nianjun , Huang Lu , Yang John , Krieger Brian , Buck Elizabeth , Wang Ruojie , Kannan Karuppiah , Chiu M. Isabel , Robinson Murray O.
Published: June 2012

The emergence of insulin-like growth factor-1 receptor IGF1R as an important cancer target is evidenced by the growing number of clinical trials currently underway for both small molecule inhibitors and antibody therapeutics developed against it. In preclinical studies, perturbation in IGF1R signaling has been associated with tumor growth inhibition, blockade of metastasis, and enhanced sensitivity to therapeutic agents, including cytotoxic chemotherapeutics and molecular targeted agents. Currently agents directed against IGF1R are being evaluated in clinical trials for their therapeutic utility.

Given the differential dependence on IGF1R exhibited by human tumors and xenograft lines, there is a need for additional preclinical models in which the relationship between genetic context and drug response in solid tumors can be better understood in an in vivo setting. To this end, we utilized our HER2 driven inducible breast cancer model to create a panel of genetically-engineered mouse tumors, each driven by the human IGF1R. Some of the models were also engineered to express a cognate ligand IGF2. In these models, tumors initially driven by human HER2 expression are re-programmed by the addition of the human IGF1R cDNA upon withdrawal of the original HER2 expression. Thus, the newly introduced IGF1R- IGF2 cDNA functionally maintains tumor survival by re-directing downstream signalling effectors in a process referred to as ‘Directed Complementation’ (DC). Using this approach, introduction of IGF1R strongly complemented tumor formation across the panel, whereas introduction of the ligands, either IGF1 or IGF2, alone or in combination did not. We recently showed that treatment of IGF1R/IGF2 DC tumors with OSI-906, a potent, selective orally active inhibitor of IGF1R, resulted in tumor regression within a week of treatment, with corresponding loss of phospho-IGF1R signaling (AACR 2009: Abstract # 2902, Lerner, et al). These results demonstrate the singular dependence of these tumors on IGF1R signaling, as well as the in vivo efficacy of the compound.

Next, we sought to extend the utility of these validated tumor models to drug discovery by establishing IGF1R-dependent cell lines for high-throughput screening.

Here we describe detailed molecular characterization of these cell lines derived from the IGF1R/IGF2 DC tumor models as well as their drug sensitivities. Importantly, we show that the IGF1R/IGF2 DC cell lines, like their parent tumors, retain activation of the pathway as well as sensitivity to OSI-906. Therefore, these tumor models and derived cell lines provide an effective preclinical tool for the development of targeted anti-IGF1R therapies.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A233.
Abstract A254: A phase 1 safety and pharmacokinetic (PK) study of PI3K/TORC1/TORC2 inhibitor, XL765 (SAR245409), in combination with erlotinib in patients (pts) with advanced solid tumors
Authors: Janne Pasi A. , Felip Enriqueta , Cedrés Susana , Cohen Steven J. , Engelman Jeffrey A. , Lucca Joan , Martínez Pablo , von Mehren Margaret , Gendreau Steven B. , Lara Katherine K. , Martini Jean-Francois , Cohen Roger B.
Published: June 2012

Background: XL765 is an oral, selective inhibitor of Class I PI3Ks, as well as TORC1 and TORC2. The PI3K pathway is frequently dysregulated in cancer cells and has been implicated as a mediator of resistance to EGFR inhibitors. In preclinical studies, XL765 has demonstrated dose-dependent target modulation in tumor xenograft models. In an ongoing Phase 1 single agent clinical study, XL765 has exhibited robust pharmacodynamic activity in diverse solid tumors.

Methods: Pts with advanced solid tumors are enrolled in successive cohorts of 3 to receive escalating doses of XL765 in combination with erlotinib. Cycles consist of 28 days of XL765 and erlotinib, each given qd. During the ongoing dose-escalation phase of the study there is a 14-day run-in period of erlotinib monotherapy. Cycle 1 safety data determine dose limiting toxicities (DLTs). Tumor response is evaluated every 8 weeks.

Results: As of 24Aug2009, 14 pts with advanced solid tumors have been enrolled: NSCLC (11), CRC (1), liposarcoma (1), and endometrial adenocarcinoma (1). Most of the NSCLC pts have been previously treated with erlotinib before entering the study. Pts have been treated at 2 dose levels of XL765 (30 and 50 mg) administered once-daily in combination with 100 mg erlotinib. No DLTs or study treatment-related SAEs have been reported. As of 03Aug2009, AEs have been reported for 7 pts. Grade 3 adverse events of nausea, vomiting, and anorexia considered related to study treatment were observed in one pt dosed at 30 mg qd. The PK of XL765 given in combination with erlotinib is consistent with that observed for XL765 given alone (Exelixis study XL765-001). Likewise, XL765 does not appear to alter the PK of erlotinib. In skin biopsies, substantial post-dose reductions were evident in phosphorylation of AKT, the mTOR substrate 4EBP1, and EGFR. For example, progressive pharmacodynamic effects were evident in serial skin biopsies from a pt with EGFR-mutant NSCLC previously treated with erlotinib who has remained on study through 26 weeks, culminating in decreases in pAKT-T308 (72%), p4EBP1 (57%), pERK (73%), and pEGFR (50%) at Day 85 post-start of combination dosing (50 mg XL765/100 mg erlotinib). Pharmacodynamic analyses of paired tumor biopsies are in progress. Of the first 9 patients enrolled, five have remained on study for at least 12 weeks.

Conclusions: XL765 in combination with erlotinib is generally well tolerated at doses up to 50 mg XL765/100 mg erlotinib, with no apparent drug-drug PK interaction, and results in robust simultaneous inhibition of PI3K and EGFR signaling.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A254.
Abstract A257: Antitumor activity of E-3810, a new, potent and selective dual inhibitor of VEGF and FGF receptors
Authors: Damia Giovanna , Bello Ezia , Colella Gennaro , Scarlato Valentina , Giavazzi Raffaella , D'Incalci Maurizio , Cereda Roberta , Cavalletti Ennio , Spinelli Silvano , Camboni Gabriella
Published: June 2012

E-3810, 6-[[7-[(1-aminocyclopropyl)methoxy]-6-methoxy-4-quinolyl]oxy]-N-methyl-naphthalene-1-carboxamide, is a novel small molecule that potently and selectively inhibits VEGFR1-3 and FGFR-1 tyrosine kinases (TKs) with IC50 in the 10-20 nM range. Concentrations at least 1-order higher were required to inhibit FGFR-2 and 3, PDGFRα and β and c-Kit receptors and no relevant activity was seen against different cancer-related TKs. E-3810 1-10 nM inhibited the ligand-dependent phosphorylation of VEGFR-2 and FGFR-1 and the downstream phosphorylation of Erk in HUVEC cells; inhibition of PDGFR- auto-phosphorylation in NIH-3T3 cells was detected at concentrations higher than 100 nM. E-3810 inhibited the VEGF- and FGF-driven growth of HUVEC cells (IC50 40-50 nM), while anti-proliferative activity on different human cancer cell lines was shown at 10-30 M concentrations. E-3810 was tested in different human tumor xenografts in nude mice, including colon HT-29, ovarian A-2780, renal A498, RXF393 and SKN12I carcinomas. A dose dependent antitumor effect was observed in a range from 10 to 40 mg/kg given orally for 30 consecutive days. The optimal dose schedule, 20 mg/kg for 30 days, was well tolerated and caused up to 90% suppression of tumor growth. Sustained growth inhibition was also seen when treatment was repeated after a 2-week dosing interval or when dosing was initiated at a larger tumor volume (average 400 mm3). Tumor regression was observed in the RXF393 xenograft. E-3810 showed antitumor activity equal or better than Sunitinib and Brivanib at their respective optimal doses. Pharmacokinetic studies in mice demonstrated high oral bioavailability, high volume of distribution and terminal half life of 4 hrs. After single and repeated administration E-3810 systemic exposure was proportional to the dose, without any accumulation after repeated doses. The good bioavailability and the remarkable antitumor activity of E-3810 in several tumor xenografts make this compound an interesting candidate for clinical development.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A257.