bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A1: The physiological form of MetAP2 can be inhibited through binding to either of the two active-site metals
Authors: Wallis Nicola G. , Berdini Valerio , Besong Gilbert , Chessari Gianni , Coyle Joe , Graham Brent , Madin Andrew , O'Brien Alistair , Richardson Caroline J. , Smith Kirsten , Thompson Neil T. , Vinkovic Mladen , Williams Pamela A.
Published: June 2012

Methionine aminopeptidases (MetAP) are metalloenzymes that remove the N-terminal initiator methionine from newly synthesized polypeptides allowing essential post-translational modifications such as acetylation and myristoylation to take place. MetAP2, one of the two eukaryotic forms of the enzyme, was identified as the target of fumagillin, a natural product with anti-angiogenic properties that inhibits the proliferation of endothelial cells. Clinical activity has been seen for a semi-synthetic analogue of fumagillin, TNP470, suggesting MetAP2 is a good target for inhibiting angiogenesis. In vitro, MetAP2 appears to have sites for two divalent metal ions within its active site but there has been much discussion around the identity and number of metal ions actually present in the physiological states of the various MetAPs. An understanding of the physiologically relevant metalloform of the enzyme is essential for designing inhibitors that are active in cells. We have used tool compounds that bind the active site metals in diverse ways to investigate the relevance of the two potential metal binding sites in MetAP2.

Using our fragment-based screening approach, Pyramid™, we screened the manganese-form of the MetAP2 enzyme. We identified multiple low-molecular weight fragment hits and confirmed their modes of binding to the two metals in the active site of MetAP2 by X-ray crystallography. Three hit series, which bound metal 1 only, metal 2 only or both metals 1 and 2 were chosen for further optimization using structure-based drug design. Optimized lead compounds had potent inhibitory activity against the in vitro MetAP2 enzyme (∼100 nM) and in HUVEC proliferation assays, whilst also showing greater than 1000-fold selectivity for MetAP2 over MetAP1. Examples from each series, representing different active site metal binding modes, were used as tool compounds to investigate the mechanism of action in cells. The levels of the MetAP2 substrate, 14-3-3, were monitored by western blot in HUVECs treated with these compounds. Levels of methionylated 14-3-3 increased upon treatment with compounds from each of our series indicating the substrate was not being processed and that in each case the compound was inhibiting MetAP2 in these cells.

These data indicate that the physiological form of MetAP2 can be inhibited by compounds which bind solely to either of the two active-site metals, suggesting that both metals must be present in the intra-cellular form of MetAP2 and allowing multiple approaches to inhibiting this key angiogenic target. The lead series identified here provide chemically diverse scaffolds for further optimization of drug like properties.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A1.
Abstract A10: Antiangiogenic therapy (VEGF-R1–3 and PDGFR inhibitor) increases tumor hemoglobin saturation and decreases interstitial pressure, and microvessel density
Authors: Hanna Gabi , Tailor Tina , Palmer Greg , Herbert Joseph , Vlahovic Gordana , Dewhirst Mark
Published: June 2012

Methods: We evaluate the effects of pazopanib, a novel multi-targeted tyrosine kinase inhibitor, by examining its effects on tumor oxygenation, interstitial pressure (IFP), and vascularity as compared to an untreated control group.

Nude mice (n=35) were injected with A-549 NSCLC tumor cells. Tumors reached an average volume of 650 mm3 before treatment with pazobanib 100mg/kg/day, Pazobanib 30mg/kg/day. or Vehicle (Control). Animals were treated daily for 14 days.

Spectroscopic Measurements: A novel Skinskan spectrofluorometer was used to obtain non-invasive measurements of hemoglobin saturation (Hbsat) and total Hb for each tumor. Measurements were performed every other day, starting the day of treatment. These values were analyzed by a custom MATLAB program.

IFP Measurements: IFP was measured on the last day of treatment(day 14), prior to sacrifice.

Immunohistochemical staining: The number of vessels, determined by positive CD31 staining, was averaged over 6–8 image fields to obtain microvessel density (MVD).

Results:

Optical Spectroscopy: A significant increase in Hbsat was observed at day 8 for the low and high pazopanib dose groups, compared to control (p=0.0109, p=0.0290, respectively). After the 8th treatment day, Hbsat trended downward toward baseline.

Tumor IFP: Tumor IFP for animals treated with pazopanib 100mg/kg was significantly reduced, compared to control (p=0.0026). Treatment with pazopanib 30mg/kg also demonstrated a strong trend towards IFP reduction, compared to control (p=0.0645).

MVD: MVD was significantly reduced in mice treated with low and high dose pazopanib, compared to control (p=0.0271, p=0.0107, respectively).

Conclusions: Treatment with pazopanib lead to a significant reduction in IFP, MVD, and increase in tumor Hb sat. This result demonstrates that pazopanib enhances efficiency of tumor perfusion which could lead to better drug distribution into the tumor, and increased of tumor Hb saturation would correlate with better oxygenation and predicts better response to radiotherapy.

Work supported by a grant from GlaxoSmithKline.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A10.
Abstract A110: A phase 1 study of PF-04929113 (SNX5422), an orally bioavailable heat shock protein 90 (Hsp90) inhibitor administered twice weekly in patients with refractory solid tumor malignancies and lymphoma
Authors: Giaccone Giuseppe , Rajan Arun , Crandon Sonja , Gutierrez Martin , Kummar Shivaani , Jain Lokesh , Figg William D. , Houk Brett E. , Shnaidman Michael , Skillings Jamey
Published: June 2012

Introduction: Hsp90 inhibitors have the potential for broad anti-cancer effects based on the spectrum of known client proteins and increased effect on mutations that drive malignant transformation and cancer growth. PF-04929113 (SNX5422; 113) is a novel, oral, non-antibiotic, selective Hsp90 inhibitor prodrug, with preclinical anti-tumor activity in multiple tumor models. The objective of this study was to evaluate the tolerability and describe the PK of 113 and its active moiety (PF- 04928473 /SNX2112; 473) in patients with refractory solid tumor malignancies and lymphoma.

Methods: Twenty-six subjects have been enrolled to date in this Phase 1, open-label, dose-escalation study. Subjects were treated with oral 113 twice weekly without interruption at doses ranging from 4 to 100 mg/m2. PBMCs were isolated and collected at baseline and various time points after-treatment. Cell lysates from PBMCs were assayed for Hsp70 and AKT protein level using Meso Scale Discovery (MSD) technology. Serial plasma concentrations of 473 were measured by LC-MS/MS after the first dose (Day 1) and after 6 doses (Day 18). PK parameters (mean ± standard deviation) were calculated using non-compartmental methods in WinNonlin v5.0.

Results: MTD for this schedule has not yet been reached. So far it has been well tolerated. Related adverse events were mainly low grade (most frequent nausea 31%) with grade ≥ 3 were reported in 8% (diarrhea and arthritis, 1 patient each). Based on results of interim pharmacokinetic analysis for doses 4 to 44 mg/m2, median time to 473 peak plasma concentrations was 2 h (range 0.33–4 h) and median half-life was ∼8.5h both at days 1 and 18. The median (range) CL/Fm at day 1 and day 18 were, 24.7 (12.9–81.2) L/hr and 22.6 (8.6–55.6) L/hr, respectively and was consistent across the dose range. Hsp70 was elevated with drug treatment, and further elevations were noted with increasing weekly dose. Although to date, no patients have achieved PR, several have experienced prolonged stable disease including medullary thyroid cancer (>450 days), lymphoma (>150 days), and esophagus, colon, adenoid cystic, lung carcinoid, and peritoneal mesothelioma (>100 days).

Conclusions: This schedule is well tolerated with no significant drug accumulation, few severe toxicities and potential for efficacy based on prolonged stable disease. Maximal concentrations of 113 were observed 2 h after oral administration of prodrug 113. Concentrations achieved were consistent with the preclinical activity. Accrual to the study to establish MTD is ongoing.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A110.
Abstract A113: Involvement of heme oxygenase-1 (HO-1) in the differentiation of human promyelocytic leukemic cells by DMSO
Authors: Kim Jong-Suk , Noh Eun-Mi , Lee Young-Rae , Park Jinny
Published: June 2012

Heme oxygenase-1 (HO-1), an inducible enzyme with broad tissue expression, is up-regulated in response to hematopoietic stress and preserves vascular homeostasis. We investigated the involvement of HO-1 in the differentiation of HL-60 cells. Dimethylsulfoxide (DMSO), a representative differentiation inducer of HL-60 cells, induced completely decrease HO-1 expression in a time-dependent manner, but increase CD11b, indicating that HO-1 might have negative function in DMSO-induced differentiation of HL-60 cells. Zinc protoporphyrin (ZnPP), a strong inhibitor of HO-1, induced differentiation of HL-60 cells, as evidenced by a marked increase in the expression of CD11b following ZnPP treatment. In contrast, treatment with cobalt protoporphyrin (CoPP), an activator of HO-1, decreased CD11b expression. ZnPP induced down-regulation of HO-1 protein expression in HL-60 cells, while CoPP induced up-regulation. In addition, ZnPP treatment caused a decrease in pRb and cyclin D1 expression and an increase in p21 and p27 expression. These results suggest that down-regulation of HO-1 might be necessary for DMSO-induced HL-60 differentiation. This study provides the first evidence that HO-1 plays an important role in DMSO-induced differentiation of HL-60 cells.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A113.
Abstract A116: Radio- and chemosensitization through inhibition of DNA-PK by KU-0060648
Authors: Bardos Julia , Shea Kerry , Parveen Nahida , Peacock Marcus , Deacon William , Cranston Aaron , Albertella Mark , Menear Keith , Smith Graeme , Martin Niall
Published: June 2012

DNA double strand breaks (DSBs) are profoundly cytotoxic unless rapidly repaired. Non-homologous end joining (NHEJ) is the predominant pathway of DSB repair in mammalian cells. DNA-dependent protein kinase (DNA-PK), a member of the PI3-kinase like kinase (PIKK) family, is an essential component of NHEJ, and DNA-PK inhibition in combination with radio- or chemotherapy therefore represents an attractive therapeutic approach in cancer therapy. Through medicinal chemistry, a series of DNA-PK inhibitors of increasing potency was identified, resulting in KU-0060648, a potent DNA-PK inhibitor with an IC50 value of 10nM against the enzyme and high selectivity for DNA-PK over other PIKKs and a panel of 60 kinases.

In cellular assays, KU-0060648 significantly increases cell death induced by ionising radiation (IR) or chemotherapeutic treatments, such as doxorubicin or etoposide. KU-0060648 does not sensitise DNA-PK-deficient cells to IR or chemotherapeutic treatments, further highlighting its selective nature. The compound was also found to preferentially kill DNA repair-defective cells in combination with IR or chemotherapy, indicating a therapeutic advantage for this class of agent in DNA repair-compromised tumor cells.

Auto-phosphorylation of DNA-PK at serine 2056 is inhibited by KU-0060648, and significant changes in IR- or chemotherapy-induced H2AX- and KAP-1 phosphorylation in the presence KU-0060648 over time clearly indicates inhibition of DNA damage repair by this compound. Pharmacokinetic and pharmacodynamic profiling of KU-0060648 revealed that the compound has very good oral bioavailability and a favourable half life. In a fractionated IR xenograft model, dosing with KU-0060648 caused significant tumor growth delay compared to the control group. PK/PD relationships were established to define the exposures and level of DNA-PK inhibition required for effective radiosensitisation. Our results to date support further in vitro and in vivo evaluation of this novel class of therapeutic agents as a potential clinical radio- and chemosensitiser.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A116.
Abstract A117: Chk1-dependent up-regulation of ribonucleotide reductase R2 in response to camptothecin-induced DNA damage
Authors: Zhang Yong-Wei , Jones Tamara , Martin Scott , Caplen Natasha , Pommier Yves
Published: June 2012

To investigate drug mechanisms of action and identify molecular targets for the development of rational drug combinations, we are conducting synthetic siRNA-based RNAi screens to identify genes whose silencing affects anti-cancer drug responses. One such screen indicated that the silencing of RRM1 and RRM2, which encode the large and small subunits of the human ribonucleotide reductase (RNR) complex respectively, markedly enhances the cytotoxicity of the Topoisomerase 1 (Top1) inhibitor camptothecin (CPT). Silencing of RRM2 was also found to enhance DNA damage as measured by -H2AX. Further studies showed that CPT up-regulates both RRM1 and RRM2 mRNA and protein levels, and induces the nuclear translocation of RRM2. The Checkpoint kinase 1 (Chk1) was also up-regulated and activated in response to CPT. CHEK1 down-regulation by siRNA and small molecule inhibition of Chk1 activity blocked induction of RRM2 by CPT. CHEK1 siRNA also suppressed E2F1 up-regulation by CPT, and silencing of E2F1 suppressed the up-regulation of RRM2. Silencing of ATR or ATM, and inhibition of ATM activity by KU-55933 blocked Chk1 activation and RRM2 up-regulation. This study links the function of known components of CPT-induced DNA damage response with proteins required for the synthesis of dNTPs, presumably in preparation for DNA repair. Specifically, we propose that, upon DNA damage, Chk1 activation, mediated by ATM and ATR, regulates RRM2 expression through the E2F1 transcription factor. Up-regulation in RRM2 levels, coupled with its nuclear recruitment suggests an active role for RNR in the cellular response to CPT-mediated DNA damage that could potentially be exploited as strategy for enhancing the efficacy of Top 1inhibitors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A117.
Abstract A118: The PARP1 inhibitor olaparib (AZD2281) enhances radiation-induced DNA damage in BRCA1/BRCA2 wild-type head and neck squamous cell carcinoma cell lines
Authors: Frederick Barbara A. , Fletcher Ashley , Raben David
Published: June 2012

Introduction: In 2008, there were approximately 65,000 cases of head and neck cancer diagnosed in the United States, with over 500,000 cases worldwide. For many patients with locally advanced disease, multiple treatment modalities are combined and typically include radiotherapy, which induces DNA damage in tumor cells. Poly (ADP-ribose) polymerase 1 (PARP1) is involved in maintenance of genome integrity, and functions as a key mediator of DNA repair. The objective of this study was to determine if combining the PARP1 inhibitor olaparib (AZD2281) with radiation in head and neck squamous cell carcinoma (HNSCC) cell lines results in enhanced DNA damage and cell death.

Materials and Methods: The effects of olaparib and radiation were assessed in the BRCA1/BRCA2 wild-type HNSCC cell lines UMSCC2, HN31, MDA-584, and UMSCC25. DNA damage following treatment was determined 2 hours post-radiation by immunofluorescence and flow cytometry using γ-H2A.X-Alexa488 conjugated antibodies. Single cell electrophoresis (comet assay) 24 hours post-radiation was used to measure DNA fragmentation, and long-term cell survival was evaluated by clonogenic survival.

Results: In all cell lines tested, both radiation and olaparib alone induced DNA damage as measured by γ-H2A.X foci formation, and the combination of olaparib and radiation significantly enhanced this response. DNA fragmentation was also greater in combination treatments, and this lead to a lower clonogenic survival for all cell lines.

Conclusions. The combination the PARP inhibitor olaparib with radiation enhanced DNA damage in HNSCC cell lines and lead to greater cell death. This study demonstrated that PARP1 inhibition combined with a DNA damaging agent such as radiation was effective in inhibiting growth of BRCA1/BRCA2 wild-type cell lines.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A118.
Abstract A119: A proteomics-based approach to identify biomarker profiles of homologous recombination deficiency (HRD) in breast cancer cell lines
Authors: Prime John E. , Unwin Louise , Hunter Jill , Allum Richard , Nickson Janice A. , Rowlinson Rachel , Giavara Sabrina , Lueck Carsten , Lau Alan , O'Connor Mark J.
Published: June 2012

Background: The oral PARP inhibitor olaparib (AZD2281, KU-0059436) has been shown to have single agent clinical activity in tumors from patients with hereditary BRCA mutations (Fong et al., 2009). Pre-clinical studies have also shown sensitivity to PARP inhibition in cells deficient in non-BRCA components of the homologous recombination (HR) DNA repair and signalling pathways. These include proteins such as NBS1, ATM and CHK2 (McCabe et al., 2006), suggesting the broader clinical potential of PARP inhibitors in tumors that are HR deficient (HRD). Thus the ability to identify tumors with HRD will be critical to realising the full potential of this targeted therapy. A proteomics study analysing isogenic breast cancer cell lines in which key HR gene expression had been stably knocked down was performed to identify a broad protein biomarker profile which may contribute to this patient selection strategy. This study also offered the opportunity to identify novel HR associated proteins and a greater understanding of DNA damage responses.

Methods: Nuclear or phospho-protein enriched samples from stable RNAi knockdown breast cancer cell lines (CAL51 and/or MCF7) for 6 key HR genes (BRCA1, BRCA2, ATM, CHEK2, MRE11, NBS1) were analysed using 2 dimensional-difference gel electrophoresis (2D-DIGE) in comparison with parental wild type and/or non specific control cells. The identities of proteins of interest were determined by mass spectrometry. Further analysis of these results was undertaken through bioinformatics approaches utilising the Ingenuity Pathway Analysis (IPA) and GeneGo software tools.

Results: Significant differences in protein abundance were observed in 308 proteins through these proteomic analyses. In depth bioinformatics analysis of these results determined their functional roles, their relationship to the double strand break repair (DSBR) pathways such as HR and non homologous end joining (NHEJ) and DNA damage response (DDR) signalling through network modelling. Where no direct relationship was apparent for these putative HRD associated proteins, their linkage was explored back to the specific HR gene knocked down in the cell line that gave the protein abundance change result. The most consistent cellular functions and pathways associated with the protein changes across the different gene knock downs included cell death, post-translational modification and protein folding.

Conclusions: The abrogation of key genes in the HR pathway leads to wide ranging effects on downstream cellular functions including cell death, post-translational modification and protein folding. These candidate biomarkers of HRD are being developed into a biomarker profile to predict response to olaparib in the clinic.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A119.
Abstract A120: Cellular inhibition of Chk2 kinase and potentiation of camptothecins and radiation by the novel Chk2 inhibitor PV1019
Authors: Jobson Andrew , Lountos George T. , Lorenzi Philip , Connelly John , Tropea Joseph E. , Zoppoli Gabriele , Kondapaka Sudhir , Zhang Guangtao , Caplen Natasha J. , Cardellina John H. , Yoo Stephen S. , Monks Anne , Self Christopher , Waugh David S. , Shoemaker Robert H. , Pommier Yves
Published: June 2012

Chk2 is a checkpoint kinase involved in the ATM pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported NSC 109555 as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039), which is a selective sub-micromolar inhibitor of Chk2 in vitro. The co-crystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against IR-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 as well as Chk2 siRNA can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A120.
Abstract A141: Synthesis and bioactivity of core-expanded calixarene-based antitumor agents
Authors: Levine Joseph I. , Dings Ruud P.M. , Loren Melissa L. , Hoye Thomas R. , Mayo Kevin H.
Published: June 2012

Anginex, a synthetic 33mer β-sheet-forming peptide, is a potent inhibitor of angiogenesis in vitro and in vivo. Previous studies demonstrate that Anginex, through binding to one of the hydrophobic faces of human galectin-1 (hGal-1), prevents endothelial cell adhesion to the extracellular matrix, induces apoptosis in angiogenically-activated endothelial cells (ECs), and suppresses tumor growth in animal models. Since small molecules have several pharmacologic advantages over therapeutic peptides, we sought to design a small molecule peptidomimetic of anginex. Calix[4]arene was chosen as the basic scaffold to mimic the approximate molecular dimensions, amphipathicity, and cationic topology of Anginex. Chemical modification of the hydrophobic (upper rim) and hydrophilic (lower rim) faces of calix[4]arene resulted in the discovery of a lead compound, 0118, possessing in vitro and in vivo activities similar to that of anginex. These studies reveal that small hydrophobic groups on the upper rim and dimethylaminoethylacetamide (DMAEA) moieties on the lower rim of 0118 are important for its biologic activity.

Until now, structure-activity relationship (SAR) development of calixarene has focused on modifications of its hydrophobic and hydrophilic surfaces. Since the hGal-1-Anginex binding site is a hydrophobic surface, we hypothesize that a larger hydrophobic calixarene surface area will translate into enhanced bioactivity. We have chosen calix[6]arene and tetrathiacalix[4]arene as tools to explore the core-expanded SAR of our lead compound. A calix[6]arene analog of 0118 not only has a larger hydrophobic footprint, but also a greater intra-annular topoisomerization rate which should allow for greater conformational space sampling and, therefore, better inducible fit within its molecular receptor. The tetrathiacalix[4]arene derivative, with its longer C-S bonds (relative to the methylene bonds of calix[4]arene and calix[6]arene), should allow for a hydrophobic footprint larger than the parent 0118 but smaller than that of the calix[6]arene analog. The current studies focus on the synthesis and biologic activity of these 0118 core-expanded derivatives.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A141.
Abstract A144: TriN 2755: Antitumor activity of a new cytotoxic therapeutic of the triazene class
Authors: Bausch Niko , Baltes Steffen , Fiebig Heinz-Herbert , Beckers Thomas , Barth Wolfgang
Published: June 2012

The new cytotoxic anti-neoplastic agent TriN 2755, containing a triazene unit with alkylating properties, has been tested in a variety of patient-derived tumor xenograft models. In these models, TriN 2755 displays potent in vivo anti-tumor activity. In rat, dog and mini pig toxicity studies, TriN 2755 demonstrated a favourable safety profile. Here, we present efficacy data in nude mice of TriN 2755 tested in 15 human tumor xenograft models derived from 9 histologies

Oncotest has developed >200 patient-derived solid tumor models growing s.c. in nude mice. These human tumors were transplanted directly into mice in order to retain the malignant phenotypes as in the patient. Since tumor morphology and sensitivity to anti-cancer drugs is being preserved in these models, results are of high clinical relevance.

A total of 15 xenograft models, among them 4 mammary, 3 melanoma, 2 prostate, 1 colon, pancreas, lung, head and neck, uterine body and bladder tumor models were tested. Tumor-bearing nude mice received TriN 2755 i.p. at doses of 300 to 540mg/kg and different treatment schedules. Treatment started when tumors had grown to diameters of 6–8 mm. Anti-tumor activity was assessed as tumor volume inhibition relative to a vehicle control group (T/C value in %). Tolerability was analysed by recording mortality and body weight loss.

TriN 2755 is a promising anti-cancer agent with an average optimum T/C value of 29.7 % over the 15 experiments representing each tumor model. Optimum T/C values below 50 % were reached in 9/15 tumor models, borderline activity with optimum T/C between 50 and 65 % was observed in 4/15 xenografts with only 2 xenografts being resistant to TriN 2755 (optimum T/C value above 65 %). TriN 2755 had a long lasting therapeutic effect in the melanoma MEXF 462, colon adenocarcinoma CXF 280 and mammary MAXF 401 tumor models. On the last day of treatment, most tumors were either in partial or complete remission. This remission generally lasted until the end of the observation period (days 45, 56 and 42, respectively). TriN 2755 was more effective in these studies than standard-of-care drugs tested in parallel (5- Fluorouracil in colon cancer and Temozolomide in melanomas). Tolerability of TriN 2755 was good with a moderate mortality rate and body weight loss.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A144.
Abstract A147: Novel fluorinated taxane SB-T-12854 active in drug-resistant and sensitive cell lines: Human, pig, rat metabolism, cell transport, and effects on cell cycle
Authors: Gut Ivan , Ehrlichova Marie , Vaclavikova Radka , Ojima Iwao , Simek Petr
Published: June 2012

SB-T-12854 is a novel taxane where fluorine in the molecule could decrease metabolism and increase activity over paclitaxel with efficiency limited by multidrug resistance due to ABCB1 transport, metabolism, and other factors.

Purposes of the study: this study investigated metabolism of SB-T-12854 by human, pig and rat CYP enzymes, cytotoxicity of the drug and isolated metabolites, transport in drug-resistant NCI/ADR-RES cells and sensitive MDA-MB-435, and effects on the cell cycle.

Experimental procedures: Metabolism of SB-T-12854 was studied in human, pig, rat and CYP3A1/2-induced rat microsomes and human cDNA-expressed CYP enzymes in vitro. The metabolites were determined by HPLC and MS analyses.

Human tumor lines MDA-MB-435 and NCI-ADR-RES (NCI, Frederick MD) were kept in standard conditions. Viability was assessed by the MTT assay. Transport of SB-T-12854 by tumor cells was determined by measuring their cellular levels by HPLC. Progress in the cell cycle was determined by FACS.

Summary of new and unpublished data: In human, pig and rat microsomes SB-T-12854 formed the same four products at similar rates, but CYP3A1/2-induced rat microsomes formed 13 metabolites at 16-fold higher rate indicating very high activity of CYP3A, the main human CYP form of varied expression. The cDNA-expressed CYP3A4 formed the same products, but CYP1A2, 1B1, 2A6, 2C9, or 2E1 were inactive. The time course of metabolism and further metabolism of isolated metabolites showed their mutual conversion and identified primary and secondary products. In rat microsomes, Km values for metabolites M5-6 and M9-12 were 14.01 and 6.89 (M), respectively, and Vmax (pmol/min/mg protein) were 70.4 (M5-6) and 370.4 (M9-12).

In MDA-MB-435 cells some metabolites were inactive, but others were considerably more cytotoxic than SB-T-12854 itself in contrast to paclitaxel whose metabolites are less active. SB-T-12854 was 6-fold more cytotoxic than paclitaxel in MDA-MB-435 cells and 30-fold more potent in NCI/ADRRES cells. In both cell lines SB-T-12854 caused G2/M block in the same way as paclitaxel.

Conclusions: SB-T-12854 is metabolized by human, pig and rat CYP3A enzymes to identical and to some markedly more active products in contrast to paclitaxel, which is only inactivated. SB-T-12854 is 30-fold more cytotoxic than paclitaxel in ABCB1 highly expressing NCI/ADR-RES cells, partly because SB-T-12854 is not transported by ABCB1 in contrast to paclitaxel. SB-T-12854 influenced the cell cycle of NCI/ADR-RES and MDA-MB-435 cells in the same way as paclitaxel causing G2/M block, but at substantially lower concentrations. Lack of ABCB1 transport and higher cytotoxicity suggest a potential advantage of this taxane over paclitaxel, which is ineffective against certain tumors, due to high ABCB1 expression.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A147.
Abstract A150: Ciglitazone, PPAR agonist, induces cell death independent of peroxisome proliferator-activated receptor γ (PPARγ) in glioma
Authors: Noh YooHun , Lee Myoung Woo , Kim Dae Seong , Kim Hye Ryung , Chueh Hee Won , Lee Soo Hyun , Yoo Keon Hee , Sung Ki Woong , Jung Hye Lim , Koo Hong Hoe
Published: June 2012

Ciglitazone (CGZ), peroxisome proliferator-activated receptor γ (PPARγ) agonist, is recognized to regulate multiple signal pathways and induce apoptosis in various cancer cells. CGZ induces apoptotic cell death through binding to PPARγ, but interdependence between CGZ and PPARγ is not clear. In this study, over 30 εM CGZ observed significant cytotoxicity in T98G cell line, but not below 25 µM. Cotreatment with 20 µM CGZ and 30 µM GW9662, an antagonist of PPARγ, synergistically induced apoptotic cell death. However, treatment with 30 µM GW9662 not significantly induced cell death and cell-cycle arrest on T98G cells pre-treated with 20 µM CGZ. These results indicate that CGZ may induce cell death independent of PPARγ in T98G cells. In addition, only treatment with 20 µM CGZ in T98G cells induced the activation of Akt. However, cotreatment with 20 µM CGZ and 30 µM GW9662 gradually decreased the activation of Akt. These data indicate that CGZ enhance the antiapoptotic effects by PPARγ binding, while free CGZ on cotreatment with GW9662 PPARγ-independently activates cell death signaling pathway through Akt inactivation. We suggest that combination of CGZ and GW9662 could be therapeutic application for the treatment of glioma.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A150.
Abstract A160: Regulation of resistance of prostate cancer cells to chemotherapy by nanog, a transcription factor for self-renewal of stem cells
Authors: Wang Man-Tzu , Nie Daotai
Published: June 2012

Nanog is a transcription factor usually expressed in embryonic stem cells that plays essential role in the stem cell self-renewal. Expression of Nanog has been detected in a number of cancer cells, especially in those positive for stem/progenitor markers. In the present studies, it was found that there was elevated expression of Nanog in the surviving fractions of tumor cells after chemotherapy. Prospective enrichment of tumor cells with endogenous Nanog expression led to enrichment of tumor cells resistant to Taxol, vinblastine, and doxorubicin, but not camptothecin (CPT). The increased resistance in Nanog expressing cells was attenuated, but not abolished, by inhibitors of p-glycoprotein, the ATP-bind cassette efflux transporter B1 (ABCB1), or by knockdown of ABCB1 via small hairpin RNAs (shRNAs). Expression of ABCB1 was increased in tumor cells enriched with endogenous Nanog expression while knockdown of Nanog reduced the expression of ABCB1 and sensitized tumor cells toward chemotherapeutics. Together, our results suggest that Nanog confers tumor cells with increased resistance to chemotherapy via regulating the expression of ABC transporters.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A160.
Abstract A166: A small molecule inhibitor of Pirin that suppresses tumor cell migration
Authors: Miyazaki Isao , Simizu Siro , Osada Hiroyuki
Published: June 2012

Background: Bcl-3 was originally identified as a putative proto-oncogene which was frequently rearranged in chronic B cell lymphocytic leukemia. Bcl-3 is a member of the I B family which modulates the activities of NF- B/Rel transcription factors. Pirin is known to be bound to Bcl-3, and expression of Pirin was observed in some tumor cell lines, however, the exact roles of Pirin in tumor cells have not been identified.

Materials and Methods: Chemical array screening - Previously, we have developed a nonselective immobilization method that makes possible the introduction of a variety of small molecules to the glass slides. To obtain the small molecules binding to Pirin, we treated the chemical array with cell lysate containing the fusion protein of Pirin-red fluorescent protein. The interaction of Pirin and small molecules was detected by a fluorescence image scanner.

Results: We found the compound, designated compound A, could bind to Pirin by using chemical array screening. We could not detect any interactions of compound A with other proteins we tested. Moreover, we carried out the pull-down assay with compound A-agarose beads, and identified Pirin as compound A-binding protein from the total cell lysate, suggesting the specificity of compound A to Pirin. Recombinant Pirin could bind to recombinant ankyrin repeat domain of Bcl-3, and the interaction was interfered by the treatment with compound A. To clarify the role of Pirin in tumor cells, we treated of the cells with compound A. We found that treatment of the cultured tumor cells with compound A or Pirin-targeted siRNA resulted in suppression of migration, but not cell proliferation.

Conclusion: By use the small molecule and siRNA, it is suggested that Pirin regulates migration of the tumor cells, and the inhibitor of Pirin might be a new chemotherapeutic potential for invasive tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A166.
Abstract A169: Identification of compounds for treatment of solid tumors using spheroid screening
Authors: Fayad Walid , Fryknäs Mårten , Olofsson Maria H. , Larsson Rolf , Linder Stig T.
Published: June 2012

A primary aim of cancer therapy is to irradicate solid tumor masses. Anticancer drugs are nevertheless not systematically developed with this aim in mind - screening and mechanistic studies are usually performed using cells grown under 2-D monolayer conditions. We have developed a procedure to screen for compounds that induce apoptosis of 3-D multicellular tumor spheroids. The chemical properties of spheroid screening hits differed significantly from conventional monolayer screening hits with regard to compound hydrophobicity and polar surface area. The spheroid screen resulted in hits which were more effective than standard chemotherapeutical agents in inducing wide-spread apoptosis of tumor spheroids. Hypotheses regarding the mechanisms of action for the selected compounds were generated using the Connectivity Map (CMAP; a compendium of gene expression signatures from drug treated cell lines). One of the hits in the spheroid screen was the alkaloid thaspine from the South American tree Croton lechleri which we show to be a dual topoisomerase inhibitor. Thaspine is insensitive to overexpression of drug efflux transporters and induces apoptosis of tumor cells in vivo. Counter-screening for compounds that do not induce apoptosis of normal epithelial cells resulted in a limited set of agents, most of which were found to be novel microtubuli inhibitors. The results show that interesting anti-cancer drugs can be identified using spheroid screening.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A169.
Abstract A170: High-content screening for inhibitors of oncogenic transcription by c-Myc
Authors: Collins Tony , Wasylishen Amanda , Penn Linda , Andrews David
Published: June 2012

There is evidence that Myc partners with another molecule called TRRAP to regulate genes required for transformation. Our hypothesis is that blocking the interaction of Myc with TRRAP will selectively kill breast cancer cells. Traditional drug screening approaches to identify compounds that will break apart two interacting proteins have rarely been successful. However, our approach is to screen compounds in live breast cancer cells for those that prevent Myc and TRRAP from binding to each other. Since the cell is constantly replacing Myc, a compound that prevents it from binding TRRAP should kill even established tumors. Importantly, we previously mapped the places on Myc and TRRAP that bind them together and showed they don't involve the parts of the protein involved in other important functions. Therefore, compounds we find that prevent Myc-TRRAP binding should not be toxic to normal cells.

Methods: We have established a new assay in which Myc and TRRAP are expressed as fusion proteins to Cerulean and Citrine fluorescence proteins, respectively. We are developing two different but related assays for the interaction between the two proteins. In one we rely on proximity resulting in complementation between non-fluorescent fragments of the Cerulean fluorescence protein. In this assay heterodimerization results in increased fluorescence. In the other assay proximity results in fluorescence resonance energy transfer (FRET) between the Cerulean and Citrine fluorescence proteins. We detect FRET by fluorescence lifetime imaging (FLIM).

Results: A novel robotic microscope that can automatically perform high speed FLIM and thereby detect Myc-TRRAP binding quickly and accurately has been assembled and tested. Our automated microscope has had an environmental stage for live cell imaging fitted and tested. Both FRET standards and constructs based on the Cerulean-Myc / TRRAP-Citrine pair have been measured to validate the assay. Cell lines are being optimized for use for screening.

Conclusions: High speed FLIM can be used to detect FRET between Cerulean-Myc and TRRAP-Citrine in live cells. Using our automated microscope we can test individually the effect of large numbers of small molecules in live breast cancer cells. This will let us identify compounds that are not only effective but that get into breast cancer cells and are not toxic to normal cells.

Relevance/Impact: To stimulate the development of new drugs effective against a wide spectrum of cancers, we are identifying small drug like molecules that disrupt the function of a particularly potent cell growth gene called Myc, which is often misregulated in breast cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A170.
Abstract A176: On the search for new tools to fight tumors and cancer pain with the aid of organic synthesis: Quicker access to pancratistatins and tetrodotoxins
Authors: Nieto-García Olaia , Cagide-Fagín Fernando , Alonso Ricardo
Published: June 2012

One of the biggest hurdles in the fight against cancer is the non-selective nature of most current treatments. On the other hand, opioids-refractive cancer pain is a most important unsolved problem. Consequently, the search and development of new drugs that are tumor-killing (while non-cytotoxic to normal cells) and/or potent analgesics are major goals in cancer research. In this context, pancratistatin and tetrodotoxin, the most prominent representatives of two different families of natural products, have been identified as highly promising leads for cancer treatment and cancer pain, respectively; in fact, they are currently at the focus of some initiatives for pharmaceutical development.

Interest in pancratistatin stems from its high level of in vitro and in vivo cell growth inhibitory activity on a wide variety of human cancer cell lines, but particularly from its mode of action that, while yet unknown, is very selective: pancratistatin does not affect normal cells in clear contrast with other agents clinically used at the moment such as paclitaxel (taxol®) or VP-16.

On the other hand, the exceptional potency and selectivity of tetrodotoxin in blocking voltage-gated sodium channels is responsible for its strong analgesic activity (3000 times more potent than morphine without the secondary side effects of opioids).

Unfortunately, the limited supply of pancratistatin, tetrodotoxin and their analogues from their natural sources is seriously precluding further progress in the area; thus stretching the need for practical chemical synthesis. We have now developed novel synthetic schemes to both families of compounds based on the expeditive gram-scale assemblage of an advanced common intermediate (a polyfunctionalized cyclohexanic core) in just three steps from commercially available compounds. In this way, we have just completed the shortest synthetic access to tetrodotoxin reported to date and two different routes to pancratistatins. We are currently preparing a number of derivatives pretended to be useful as biological probes. Details will be given at the Conference.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A176.
Abstract A178: Role of tumor and host-derived HGF in drug resistance to EGFR inhibitors in EGFR activating mutation-positive lung cancer
Authors: Kakiuchi Soji , Yano Seiji , Wang Wei , Li Qi , Yamada Tadaaki , Matsumoto Kunio , Hanibuchi Masaki , Mitsudomi Tetsuya , Yatabe Yasushi , Kuramoto Takuya , Otsuka Koji , Nishioka Yasuhiko , Sone Saburo
Published: June 2012

Background: Non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR)-activating mutations (EGFRmu) responds favorably to the EGFR tyrosine kinase inhibitors (EGFR-TKIs), gefitinib or erlotinib. However, 25–30% of patients with EGFRmu show intrinsic resistance, and the responders invariably acquire resistance to EGFR-TKIs. Here, we examined the role of hepatocyte growth factor (HGF), a specific ligand of MET, in gefitinib resistance of NSCLC with EGFRmu.

Methods: NSCLC cells, PC-9 and HCC827, with EGFRmu, but not T790M second mutation in EGFR or MET amplification, were used in this study. Sensitivity to gefitinib was determined by MTT assay. Phosphorylation of MET, EGFR, ERBB3, and the PI3K/Akt pathway were examined by Western blotting. HGF expression in 20 tumors from 16 NSCLC patients with EGFRmu who were treated with gefitinib was determined by immunohistochemistry.

Results: HGF induced resistance to gefitinib by restoring Akt phosphorylation independently of ErbB3. Specific down-regulation of MET, but not ErbB3, reversed gefitinib resistance and Akt phosphorylation induced by HGF. High levels of immunoreactivity for HGF were detected in cancer cells of 3 of 3 intrinsic resistant tumors and in one of 2 tumors with acquired resistance without T790M second mutation or MET amplification, suggesting HGF-induced resistance by autocrine mechanism. The lung cancer cells also became resistant to EGFR-TKIs when co-cultured in vitro with HGF-producing fibroblasts and co-injected into SCID mice. Importantly, combined use of gefitinib plus anti-HGF antibody or the HGF antagonist, NK4 successfully overcame the fibroblast-induced EGFR-TKI resistance both in vitro and in vivo. Co-localization of fibroblasts and HGF was detected in both xenograft tumors in mouse model and lung cancer patient specimens, suggesting HGF-induced resistance by paracrine mechanism.

Conclusions: The findings indicate that HGF-mediated MET activation is a novel mechanism of intrinsic and acquired gefitinib-resistance in NSCLC with EGFRmu by both autocrine and paracrine mechanisms. Therefore, inhibition of HGF-MET signaling may be considerable strategy for more successful treatment with EGFR-TKIs.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A178.
Abstract A179: Synergistic interactions of Lapatinib and an AKT inhibitor in HER2 positive breast cancer cell lines depends on PI3K pathway status
Authors: Korkola James , Bayani Nora , Cooper Brian , Kuo Wen-Lin , Billig Jessica , Ziyad Saffiyah , Feiler Heidi , Spellman Paul , Wooster Richard , Gray Joe W.
Published: June 2012

Purpose: Lapatinib is a dual inhibitor of EGFR/HER2. Recent evidence suggests that resistance to HER2 inhibition by lapatinib may be in part due to re-activation of PI3K-AKT signaling mediated by HER3. The purpose of this study was to screen lapatinib in combination with a pan-AKT inhibitor in a panel of HER2 amplified breast cancer cell lines to determine if this dual inhibition had synergistic effects in preventing cell line growth.

Methods: Cell lines were treated in triplicate with nine two-fold serial dilutions of lapatinib, AKTi, or a combination of the two for 72 hours. Growth was measured using a cell titer glo assay (Promega) and growth inhibition (GI) was measured using standard techniques. Synergistic interactions were measured by the median effect method and reported as a combination index using the Synergy module in R.

Results: Of 11 HER2 positive cell lines tested, four showed strong evidence of synergy in more than half of the nine concentrations used. Five cell lines showed little or no synergy (and even some evidence of antagonism). The remaining two cell lines showed an intermediate response. Of the four lines with synergy, all were mutant for PIK3CA, while only one of the five lines that did not show strong synergy was mutant for PIK3CA. Interestingly, this line did show significant synergy at one dose combination, suggesting that it was likely to be more sensitive to lapatinib + AKTi than the other four lines. From microarray data, we identified two probe sets (representing one gene, SASH1) at a False Discover Rate of less than 5% that showed a significant association between expression and response. SASH1 has previously been implicated as a tumor suppressor gene in breast, although at this point, it is unclear whether it plays any functional role or is simply a marker for synergistic response to lapatinib and AKTi.

Conclusions: Our work demonstrates that a combination of lapatinib plus and AKT inhibitor may be beneficial in HER2 positive patients who also have PI3K pathway mutations. Care should be taken in screening patients prior to treatment, as the combination was found to be antagonistic in some cell lines that did not have PIK3CA mutations. SASH1 may be a useful screening tool in identifying such patients.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A179.