bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A184: A phase I study of 5-azacytidine (AZA) and erlotinib (E) for patients (pt) with advanced solid tumors
Authors: Verschraegen Claire , Muller Carolyn , Bauman Julie , Rutledge Teresa , Jones Dennie
Published: June 2012

Background: Erlotinib (E), a tyrosine kinase inhibitor of EGFR, is approved for non-small cell lung cancer. 5-Azacytidine (AZA) is approved for leukemia. AZA traps DNA methyltransferase (DMT) in a covalent complex with DNA resulting in a progressive loss of DNA methylation, at concentrations that do not cause major suppression of DNA synthesis. Hypomethylation reactivates previously quiescent genes and may restore the function of genes that control cell proliferation and differentiation, such as EGFR.

Methods: A standard 3+3 phase I study of the combination of E and AZA was planned in patients with incurable solid tumors. E was given at full dose (150 mg/day continuously). Vidaza was given intravenously every 2 weeks at a starting dose of 75 mg/m2/day ×1 day. Increments were by number of AZA days: The second cohort received 2 consecutive days of Vidaza, the third, 3 days, the fourth, 4 days. One course was 4 weeks. Standard grade 3 and 4 DLTs (CTCAE version 3) were recorded on the first course of treatment. RECIST criteria were used to determine response.

Results: 18 adult patients (pt) have been enrolled. Cohort 1: 6 pt; cohort 2, 3 pt; cohort 3, 6 pt; cohort 4, 3 pt. DLTs included eye infection (1 pt in cohort 1) and a neutropenic sepsis in cohort 3. Both cohorts were expanded. Overall AEs were: grade 3 rash, diarrhea, and hypokalemia (1 each (5%)); grade 1–2 rash (60%), nausea/vomiting (38%), diarrhea (38%), fatigue (33%), mouth sores and dysgeusia (22% each), hypokalemia (16%), transaminase increase (11%), neutropenia (5%), pneumonitis (5%), urticaria (5%). The maximum tolerated dose has not been reached at 75 mg/m2/day × 4 days every 2 weeks. Enrollment will continue until AZA 100 mg/m2/day × 4 days is reached. ORR is 5% with a clinical benefit rate of 40%, and the median number of courses given is 2 (range 1–6).

Conclusions: The combination of E and AZA is well tolerated with no synergistic toxicities. The main AEs are rash and gastrointestinal. E+AZA has modest but definite activity in patients with refractory tumors, and long disease stabilization are observed in selected patients.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A184.
Abstract A208: Comparison of expression and function of two functional RARβ isoforms in human cancer: Clinical implications for the differential splicing of the RARβ gene
Authors: Swift Catherine B. , Varela Victor A. , Graves Jason T. , Dmitrovsky Ethan , Petty W. Jeffrey
Published: June 2012

Purpose: Retinoids are derivatives of vitamin A that are used for treating and preventing certain cancers. Retinoid sensitivity in the lung is linked to expression of a specific isoform of the retinoic acid receptor β (RARβ), termed RARβ1′. This study compared the function of RARβ1′ and RARβ2 and assessed the expression of these isoforms in a panel of cancers. Experimental Design: RARβ1′ and RARβ2 were independently co-transfected into BEAS-2B human bronchial epithelial cells along with a retinoid responsive reporter plasmid. Transfectants were then treated with 4 µM all-trans retinoic acid or control and reporter activation was measured. An isoform-specific antibody for detection of RARβ1′ was also developed and validated. Using this antibody, RARβ1′ expression was evaluated by immunoblot analyses of normal lung versus lung cancer tissue specimens as well as in a panel of cancer cell lines including those of breast, colon, prostate, glioblastoma (GBM) and lung cancer origins. RARβ1′ expression was then examined in an extended panel of GBM cell lines. GBM cell lines were independently treated with 13-cis-retinoic acid, and growth assays and microarray analyses were each performed.

Results: In the absence of retinoid treatment, retinoid reporter activation was increased by co-transfection of RARβ2 (P = .02) but not RARβ1′ (P = .21) in BEAS-2B cells. When treated with the retinoid, transfection of either isoform enhanced reporter activation. RARβ1′ was noted to be expressed in normal lung tissue but absent in tumor tissue from patients with each of the common types of non-small cell lung cancer. Analysis of multiple cancer cell lines revealed RARβ1′ expression only in the GBM cell lines. No appreciable expression was detected in the breast, colon, prostate, or lung cancer cell lines. Analysis of an extended panel of GBM cell lines revealed RARβ1′ expression in all examined GBM cell lines. The examined GBM cell lines were found to be sensitive to treatment with pharmacologically achievable dosages of 13-cis retinoic acid. Microarray analyses revealed several novel candidate retinoid target genes activated in SNB-19 GBM cells.

Conclusions: RARβ1′ and RARβ2 have differing effects on ligand-independent signaling. RARβ1′ was repressed in all examined lung cancer cases and in most of the cancer cell lines studied. RARβ1′ protein was abundantly expressed in GBM cell lines which may help to explain the activity of 13-cis retinoic acid for the treatment of GBM despite the modest single agent activity of retinoids for treating many epithelial cancers. Microarray data of GBM cells have uncovered several candidate retinoid-target genes that may represent novel therapeutic targets.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A208.
Abstract A213: Potent anticancer activity in vitro and in vivo by NMS-E973, a novel synthetic inhibitor of HSP90
Authors: Fogliatto Gianpaolo , Mantegani Sergio , Amboldi Nadia , Ballinari Dario , Brasca Maria Gabriella , Caprera Francesco , Cappella Paolo , Casale Elena , De Ponti Anna , Guanci Marco , Paolucci Mauro , Sola Francesco , Valsasina Barbara , Visco Carlo , Zuccotto Fabio , Colotta Francesco , Felder Eduard , Donati Daniele , Moll Juergen , Galvani Arturo , Pesenti Enrico , Isacchi Antonella
Published: June 2012

The molecular chaperone heat shock protein 90 (HSP90) is essential for the conformational maturation and stability of a variety of proteins, including kinases, transcription factors, telomerase, and other key proteins implicated in cancer development and progression. The simultaneous block of multiple signaling pathways that is induced by Hsp90 inhibition makes this approach potentially attractive in a broad range of cancer types.

Prototype geldanamicyn-derivative HSP90 inhibitors have already shown efficacy in different types of tumors, but possess liabilities inherent to the class which may ultimately limit their development. Here we describe the in vitro and in vivo characterisation of NMS-E973, representative of a novel class of non-ansamycin Hsp90 inhibitors. NMS-E973 binds HSP90 with picomolar affinity, as revealed by Surface Plasmon Resonance analysis, whereas selectivity screening showed no significant activity against a broad panel of kinases, as well as other ATPases. NMS-E973, when profiled against a panel of 91 tumor cell lines of various tissue origins, showed widespread antiproliferative activity. On-target mechanism was confirmed in multiple cell lines by observation of dose and time dependent degradation of specific oncogenes, parallel blockade of the PI3K and RAF/MEK/ERK pathways, and upregulation of HSP70, a well-known feedback response to HSP90 inhibition. In vivo, NMS-E973 had a favourable pharmacokinetic profile, good tolerability, selective retention in tumors, and induced tumor shrinkage in multiple human xenograft models in nude mice. In the B-RAF V600E driven A375 melanoma model, for which a multi-cycle regimen was adopted, shrinkage was observed both during the first treatment cycle, as well as after successive re-challenge of larger, regrown tumor masses. In conclusion, NMS-E973 is a novel, potent non-ansamycin like inhibitor of HSP90, with efficacy and pharmacokinetic properties compatible with clinical development.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A213.
Abstract A22: Response to the triple VEGFR inhibitor tivozanib (AV-951) in KRAS and EGFR driven lung tumors developed in chimeric mouse models
Authors: Bressel Angela , Zi Tong , Jesmer Kristen , Monti Anthony , Brodeur Joelle , Rideout William , Chiu Maria Isabel , Robinson Murray O. , Heyer Joerg , Zhou Yinghui
Published: June 2012

Lung cancer is one of the most prevalent and deadly malignancies in the world, with more than 213,000 new cases diagnosed each year in the US and a five year survival rate of 15%. Activating mutations in a number of key signaling genes including KRAS, EGFR, and HER2 have been identified. Mutations in EGFR are highly correlated with sensitivity to small molecule EGFR inhibitors, although resistance tends to develop within a short period of time after initiation of treatment. Targeted inhibition of KRAS has not been successful so far, which in turn fueled efforts to identify tractable alternative therapeutic points of intervention.

Because all solid tumors are thought to require neo-vascularization, pharmacologic angiogenesis inhibition may represent an attractive therapeutic strategy for these challenging tumor settings. One attractive anti-angiogenesis agent is the ATP competitive small molecule VEGFR inhibitor tivozanib (AV-951). tivozanib exhibits picomolar inhibitory activity against all three VEGF receptors, a multi-day T1/2 in humans, and demonstrates robust clinical activity demonstrated in renal cell carcinoma, the signal tumor type for VEGF pathway inhibition.

In an effort to recapitulate the stochastic nature of human cancer in genetically engineered mouse models and to maximize their use in preclinical settings, we developed a mouse tumor model strategy involving stepwise genetic manipulation of embryonic stem (ES) cells and chimera formation to enable direct tumor induction in tissues containing both normal and engineered cells. An allelic series of lung cancer models containing EGFR, KRAS, or HER2 oncogenes demonstrated that resultant adenocarcinomas arising within normal lung tissue exhibited features of advanced malignancies.

In this study, we tested the response of KRAS and EGFR driven lung tumors to the VEGFR inhibitor tivozanib in tumor bearing chimeric mice identified by bioluminescent imaging. We demonstrated that tivozanib treatment conferred significant survival benefit to the tumor bearing mice. Consistent with previous reports that tumors become more dependent on neo-angiogenesis for survival and proliferation, we observed sensitivity to tivozanib in advanced adenocarcinomas but not hyperplasia or small adenomas. In addition, we found that upon discontinuation of tivozanib treatment, tumors quickly grew back and became less sensitive to further tivozanib treatment. These results indicate that lung cancer patients could potential benefit from anti-angiogenesis therapy, but sustained inhibition of all VEGF receptors may be important to prevent development of resistance.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A22.
Abstract A226: ImmunoPET with 89Zr-DFO-trastuzumab: Monitoring the pharmacodynamic effects on HER2/neu expression by Hsp90 inhibition in vivo
Authors: Holland Jason P. , Caldas-Lopes Eloisi , Divilov Vadim , Longo Valerie A. , Taldone Tony , Zatorska Danuta , Chiosis Gabriela , Lewis Jason S.
Published: June 2012

Objectives: The positron-emitting radionuclide 89Zr (t1/2 = 3.17 days) has been isolated in high specific-activity and was used to prepare 89Zr-radiolabeled monoclonal antibodies (mAbs) for use as immunoPET imaging agents. The work presented describes the preparation of 89Zr-DFO-trastuzumab for use as a radiotracer for delineating HER2/neu positive tumors for the non-invasive imaging of breast cancer in vivo. In addition, pharmacokinetic studies on HER2/neu expression levels in response to administration of therapeutic doses of PU-H71 (a specific inhibitor of heat-shock protein 90 [Hsp90]) were conducted.

Methods: 89Zr was prepared via the 89Y(p, n)89Zr transmutation with high radiochemical yields (1.52±0.11 mCi/ Ah) and purity (>99.99%). Trastuzumab was functionalized with the tris-hydroxamate chelate, desferrioxamine B (DFO) and radiolabeled with [89Zr]Zr-oxalate at room temperature. ImmunoPET imaging experiments in female, athymic nu/nu mice bearing sub-cutaneous BT-474 (HER2/neu positive) and/or MDA-MB-468 (HER2/neu negative) were conducted. The change in 89Zr-DFO-trastuzumab tissue uptake in response to high- and low-specific-activity formulations and co-administration of PU-H71 (a Hsp90 chaperone inhibitor) was measured by using both acute biodistribution studies and Western blot analysis.

Results: 89Zr-DFO-trastuzumab radiolabeling proceeded in high radiochemical yield and specific-activity of 2.82±0.05 mCi/mg. In vitro assays demonstrated >99% radiochemical purity with an immunoreactive fraction of 0.87±7. In vivo biodistribution experiments revealed high and specific tumor uptake after 24, 48 and 72 h (64.68 ± 13.06 %ID/g; 71.71 ± 10.35 %ID/g and 85.18±11.10 %ID/g, respectively) with retention of activity for over 120 h. Pre-treatment with PU-H71 was followed by quantitative biodistribution and 89Zr-DFO-trastuzumab immunoPET imaging. Expression levels of HER2/neu were modulated during the first 24 and 48 h post-administration (29.75 ± 4.43 %ID/g and 41.42 ± 3.64 %ID/g, respectively). By 72 h radiotracer uptake studies (73.64 ± 12.17 %ID/g) and Western blot analysis demonstrated that HER2/neu expression recovered to baseline levels.

Conclusions: The results indicate that 89Zr-DFO-trastuzumab provides quantitative and highly-specific delineation of HER2/neu positive tumors and may be used to monitor treatment with Hsp90 inhibitors. Efforts towards the “current Good Manufacturing Process” (cGMP) production and clinical translation of 89Zr-DFO-trastuzumab are underway.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A226.
Abstract A24: Establishment and characterization of multi-drug resistant HER-2/3 expressing and triple-negative breast tumor xenograft models
Authors: Moore Mitchell M. , Estrada Vanessa A. , Kelly Scott G. , Nieves Francis E. , Bruckheimer Elizabeth , Papadopoulos Kyriakos P. , Patnaik Amita , Tolcher Anthony W. , Hidalgo Manuel , Wick Michael J.
Published: June 2012

Breast cancers demonstrate marked heterogeneity in clinical presentation where classification and treatments are based on the presence or absence of several protein and genetic markers. The human epidermal growth factor-2 (HER-2) receptor is a surface-bound receptor tyrosine kinase overexpressed in some cancers, especially breast and ovarian, and is a target for several approved therapies including the α-HER-2 antibody trastuzumab and duel (EGFR/HER2) kinase inhibitor lapatinib. However, many of these cancers develop resistance to these agents leaving patients with few effective alternative treatments. Lack of validated preclinical models of HER-2 targeted drug resistance impairs drug development in this area. Furthermore, breast cancers lacking expression of HER-2, progesterone (PR), and estrogen receptors (ER-α), referred to as “triple negative (TN)”, represent a minority of diagnosed cases each year but are responsible for a disproportionate number of breast cancer deaths. Few advances have been made in the treatment of TN compared with other types of breast cancer. Thus the development of effective screening tools for promising agents in these HER2 and TN subgroups is warranted.

To address these unmet needs, we have established and characterized a cell-based, HER-2/3 expressing xenograft model in both mice and rats and a panel of cell-based and low passage human tumor models representing TN breast cancer. Chronic treatment with trastuzumab or lapatinib was ineffective in the HER 2/3-expressing model in mice. In the TN panel, cell-based models showed varying sensitivity to standard platinum or taxane treatments while the CBI-0810 low passage TN model was sensitive to liposomal doxorubicin and a cisplatin/irinotecan regimen but not the α-EGFR antibody cetuximab or mTOR inhibitor temsirolimus.

Results from these studies confirm HER-2 targeted drug resistance in this breast cancer model and benchmark its value as a screening tool for agents targeting trastuzumab/lapatinib-refractory breast cancer. In addition, data demonstrate that the TN panel is a novel and highly beneficial tool for screening potential therapies for this form of breast cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A24.
Abstract A247: Preclinical antitumor activity of the MEK inhibitor RO5068760 in human tumor xenograft models
Authors: Brian Higgins , Kolinsky Kenneth , Daouti Sherif , Rizzo Christine , Wang Huisheng , Huby Nicholas , Niu Huifeng , Packman Kathryn
Published: June 2012

RO5068760 is a representative member of a new class of potent, highly selective, non-adenosine triphosphate (ATP) competitive MEK inhibitors. In tumor cells, RO5068760 selectively blocked MAPK pathway signaling by inhibiting the phosphorylation of both ERK1/2 and MEK1/2. Human tumor cell lines bearing MAPK pathway gene mutations such as B-Raf and Ras were preferentially sensitive to RO5068760 treatment in vitro. When administered orally to tumor-bearing mice, RO5068760 demonstrated dose-dependent anti-tumor activity in a variety of human xenograft models including melanoma, colorectal, pancreatic, and non-small cell lung cancer (NSCLC). RO5068760 elicited statistically significant tumor growth inhibition in all models tested when administered once daily (qd) or twice daily (bid). At an optimal dose of 100 mg/kg bid, partial regressions (PRs) were demonstrated in 3/4 tumor models, and complete regressions (CRs) in 1/4 models tested. Antitumor efficacy was proportional with drug plasma exposure and inhibition of ERK1/2 phosphorylation, with slightly better efficacy observed with bid vs qd administration in the MiaPaCa-2 (pancreatic) and H460a (NSCLC) models. In the Lox melanoma model, animals treated at doses as low as 12.5 mg/kg bid exhibited CRs, which further supported the observation made in vitro that tumor cells bearing B-Raf mutations had the greatest sensitivity to RO5068760. Combination treatment of mice bearing MiaPaca-2 xenografts with 2/3 optimal doses (ODs) of RO5068760 and Gemcitabine significantly enhanced the antitumor activity as compared to either monotherapy. These results suggest that RO5068760 is a promising candidate for further development in the treatment of human cancers with aberrant MAPK pathway activation.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A247.
Abstract A249: Potent protein kinase inhibitors block Pim kinase-mediated increase in prostate epithelial cell migration, regulation of p27 protein half-life, and secretion of hepatocyte growth factor
Authors: Kraft Andrew S. , Cen Bo , Zemskova Marina , Beharry Zanna , Smith Charles D.
Published: June 2012

Pim-1 proto-oncogene encodes a serine/threonine protein kinase that regulates apoptosis, cell cycle progression, and transcription. The expression of this protein kinase is elevated in both prostate intraepithelial neoplasia (PIN) and prostatic adenocarcinoma, suggesting an important role for Pim-1 kinase in prostate cancer development and growth. To investigate the role of Pim-1 in controlling tumor growth we have synthesized novel benzylidene-thiazolidine2, 4-dione (J. Med. Chem. (2009) 52:74) inhibitors of this kinase. The most potent members of this chemotype have IC50s of 13 nM for Pim-1. To examine the activity of these agents in prostate cancer we have first set out to define the biochemical activity of Pim-1 in epithelial cells. We have expressed Pim-1 in a mouse prostate epithelial cell (MPECs) line that demonstrates stem cell characteristics. We find that Pim-1 expressing cells produce and secrete markedly increased levels of hepatocyte growth factor/scatter factor (HGF/SF), and this protein kinase stimulates increases in HGF/SF mRNA. Additionally, expression of Pim-1 markedly increases HGF/SF induced migration of MPECs. The contribution of Pim-1 kinase to this biochemical pathway is confirmed in murine embryonic fibroblasts (MEFs) that are deficient for all three Pim protein kinases (TKO) and evidence reduced level of HGF mRNA in TKO versus wild type MEFs. The Pim-1-stimulated migration of MPECs is inhibited by two benzylidene-thiazolidine-2, 4-diones, SMI-4a and 16a, as well as known inhibitors of the HGF receptor, c-Met. HGF treatment of MPECs induced p27 upregulation that could be inhibited by the expression of Pim-1, thus allowing cell cycle progression. Further studies showed that expression of Pim-1 did not alter the mRNA level of p27, but enhanced the cell cycle-dependent degradation and thus decreased the half-life of the p27 protein. Consistent with this finding, p27 ubiquitination assays showed that Pim-1 increases this modification in vivo. We found that the Pim-1-mediated ubiquitination is regulated by complex formation between Pim-1 and Skp2, a protein component of the SCF complex, which is known to regulate the ubiquitination and degradation of p27. Pim-1 does not affect Skp2's E3 ligase activity, but appears to inhibit the degradation of Skp2 through phosphorylation. Incubation of these cells with the Pim protein kinase inhibitor, SMI-4a, decreased Skp2 expression and increased p27 and cyclin E expression. Together our data demonstrates the complex pathway by which Pim-1 protein kinase regulates HGF/SF and p27 levels, thus controlling cell migration, proliferation, and potentially transformation. Potent Pim kinase inhibitors block these two signaling pathways thus inhibiting prostate epithelial cell migration and growth.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A249.
Abstract A31: Human prostate cancer transcriptome reveals distinct signaling pathway dysregulations associated with TMPRSS2:ERG fusion
Authors: Bhasin Manoj , Lu Bin , Arredouani M. Simo
Published: June 2012

Introduction: The gene rearrangement leading to the fusion of the TMPRSS2 and ERG genes on chromosome 21 occurs in about 50% of prostate cancer cases. TMPRSS2:ERG fusion is believed to contribute to prostate tumorigenesis in mice and has been shown to be associated with aggressive disease in patients with prostate cancer. However, the molecular mechanisms driving the clinical aggressivity of fusion-positive prostate cancer remain unknown.

Hypothesis: We postulate that dissecting the molecular events taking place in prostate tumors with or without ERG fusion might lead to the discovery of novel targets for prostate cancer therapy.

Methods: RNA was purified from benign (n=8) and malignant (n=13) human prostate specimens. Malignant specimens were previously tested for the occurrence of ERG fusion and included 6 fusion(+) and 7 fusion(−) samples. We performed gene expression profiling using cDNA microarrays, and conducted in silico analysis of gene expression data, followed by detailed canonical pathway analysis and regulatory network identification.

Results: Gene expression data revealed differential expression of 3039 genes in fusion positive cancer, while only 2386 genes were differentially expressed in fusion negative cancer. Among these genes, 1096 were differentially expressed regardless of the fusion status of the tumor. Pathway analysis performed on differentially expressed genes showed that fusion positive tumors showed the highest impact occurred on genes classified in the Nitric Oxide Signaling in the Cardiovascular System Pathway and the Cardiac β-Adrenergic Signaling Pathway, while the most impacted pathways in the fusion negative tumors were the Ephrin Receptor Signaling Pathway and the Fatty Acid Biosynthesis Pathway.

Conclusion: TMPRSS2:ERG gene fusion highlights a distinct molecular subtype of prostate cancer. A better understanding of the molecular consequences of the fusion will lead to the identification of new molecular targets for diagnosis and therapy of prostate cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A31.
Abstract A34: Conquering resistance to targeted therapies through system-based analysis of clinico-molecular information
Authors: Stein Martin A. , Brems Stefanie , Seifarth Wolfgang , Zien Alexander , Frank Oliver , Jackson David B.
Published: June 2012

While the advent of targeted therapies has promised to revolutionize the success of cancer treatment, a critical review of clinical response rates provides a sobering perspective on the challenge at hand. New levels of discovery innovation are urgently required to redress this enormous medical need. Focusing on primary imatinib resistance in chronic myeloid leukemia, we demonstrate that the systems-based modeling and analysis of clinico-molecular information from responder and non-responder patients can provide unique molecular insights into the sources of therapy resistance. By then utilizing these models to prioritize therapeutic drug space, the strategy elaborates rational multi-component therapies to potentially redress the issue. Moreover, by hypothesizing that co-medications may perturb the activity of resistance model components, we utilize prescription information to “probe” the functional network in search of key resistance associated targets. Not only does this method provide a novel approach to the analysis of patient information, it also permits the repositioning of drugs from independent therapeutic areas within predicted combinatorial regimens. Taken together, our results provide an exciting new approach to the computational analysis of clinico-molecular data, and suggest a future where rationally designed theranostic-linked combination therapies could significantly address the non-responder problem.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A34.
Abstract A35: Computer modeling of nocodazole exposure on cell cultures in vitro
Authors: Fernandez Eric , Johnson George , Orrell David , Fell David , Doak Shareen H. , Chassagnole Christophe
Published: June 2012

Nocodazole is an anti-neoplastic agent which reversibly impairs tubulin assembly, resulting in blocking cells in mitosis. At high concentration, nocodazole rapidly depolymerises microtubules in vivo, preventing cells from progressing through mitosis. At lower concentrations, it blocks cells in mitosis by stabilizing microtubules. In vitro, nocodazole is frequently used to synchronise cells, but also leads to aberrant mitoses.

Physiomics and the Institute of Life Science at Swansea University have entered a collaboration to build mathematical models of cell populations in vitro capable of predicting the effect of nocodazole on cell cycle progression, mitotic defects and chromosomal aberrations. Systems biology modelling is a useful method to predict the cell cycle and cell population outcomes which may result from inhibiting cell cycle progression to different degrees with small molecule inhibitors.

In order to simulate the effect of drugs on a cell culture, Physiomics has developed a cell population simulator called SystemCell. Each cell is an instance of an autonomous cell cycle, combined to a discrete event-based state machine, which determines its proliferation state, its ploidy and the multiple stages of apoptosis in a stochastic way. The ILS has provided experimental data on the dose-response relationship of nocodazole exposure with aberrant mitosis. These experimental results have been used as a data set for the calibration of Physiomics models. The cell population simulations were performed on the High Performance Computer available at the ILS, Swansea University.

By accurately model the effect of nocodazole, this simulation platform will be particularly useful to predict the effect and dosing of nocodazole with other antitumor agents in combination.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A35.
Abstract A6: Pazopanib combination with paclitaxel and carboplatin in patients with advanced solid tumors and gynecological cancers: Results of two phase I studies
Authors: Burris Howard , du Bois Andreas , Dowlati Afshin , Gainer Shelby , Park JungWook , Stutts Melissa , Dar Mohammed , Suttle Ben , Mitrica Ionel , Tan Antoinette R.
Published: June 2012

Background: Pazopanib (PAZ) is an oral angiogenesis inhibitor targeting VEGFR, PDGFR, and c-Kit with w/ a recommended dose of 800 mg/day as monotherapy. Preclinical activity supports combining PAZ with paclitaxel (P) and carboplatin (C). Safety, PK, and and clinical activity of PAZ with P and and C were evaluated in two Phase I studies.

Methods: Two Phase I studies were conducted using a once daily QD regimen of PAZ (200–800 mg), and once every Q21 days of C (AUC 4–6 mg·min/mL) and P (175 mg/m2) to determine the optimally tolerated regimen (OTR) MTD defined as a dose limiting toxicity (DLT) frequency of <33% in Cycle 1. Study 1 (S1) was a 3 + 3 design expanded at the OTR in patients (pts) with w/ metastatic disease treated with w/ no more than 3 prior lines of therapy. Study 2 (S2) enrolled subjects with w/ gynecological cancer in 2 cohorts of 6 pts each as first-1st line treatment.

Results: In Study 1S1, 34 pts (mean age 54; M/F: 17/17; ECOG 0/1: 19/15) were enrolled. Breast (29%) and esophagus (12%) were the most common primary tumor sites. In Study 2S2, 12 pts (mean age 53.754, M/F: 0/12; ECOG 0/1: 8/4) were enrolled. Ovary (50%) and endometrium/uterus (25%) were the most common primary tumor sites. The frequency of adverse events (AEs), regardless of causality, in the 2 studies (1 vs 2), were similar for neutropenia (82% vs 75%) and fatigue (68% vs 75%) and different for nausea (71% vs 50%), thrombocytopenia (68% vs 33%), anemia (65% vs 25%), and abdominal pain (24% vs 50%). The OTR MTD in Study 1S1 was P 175, C AUC 5, and PAZ 200; an OTR MTD was not determined for Study 2S2 due to poor tolerability. In Study 1S1, in the presence of PAZ 200 mg and 400, mg, the AUC (0–23) of C increased by 41 and 31%, respectively and while the Cmax for P increased by 43 and 40%, respectively. Best response in Study 1S1 at the OTRMTD (n=13) was CR [2 pts (15%); 406 and 446 days] and PR [3 pts (23%); 42–208 days].

Conclusions: Full doses of PAZ and C were not tolerated in combination with w/ P due predominantly to hematologicale toxicities in Study 1S1 and to non-hemeatological toxicities in Study 2S2. A drug interaction between PAZ and P and C along with more extensive prior therapy may explain increased myelotoxicity with this triplet in Study 1S1. Based on this PK interaction, lower doses of P and C may still provide adequate therapeutic exposure and better safety profile when combined with a higher dose of PAZ relative to the OTR MTD in study.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A6.
Abstract A60: Targeting brain tumor stem cells
Authors: Bourseau Erika , Lemaire Laurent , Hervouet Eric , Valette François , Menei Philippe , Berger François , Wion Didier , Benoit Jean-Pierre , Garcion Emmanuel
Published: June 2012

The brain tumor stem-cell (BTSC) hypothesis proposes with acquired self-renewal properties establish a brain tumorigenic-behavior (1). Thus, failure to cure brain cancer has been attributed to the fact that typical cytotoxic therapies target rapidly proliferating brain tumor cells, while sparing the BTSC compartment, which has a low proliferation rate and high tumorigenic potential. Taking advantage of the recent development of nanotechnologies that may offer innovative drug-delivery systems for improved efficacy, specificity and biological safety (2), the aim of our work is to develop nanoparticle-based treatments targeting BTSCs.

The first part of our work represents a comprehensive study of the aggressiveness of the BTSC was shown to be associated with the expression of the neural precursor cell marker AC133. Hence, the percentage of AC133 expressing glioma cells is increased after maintenance or exposure to moderately low oxygen levels (3). Our data indicate that cells maintained at 3% oxygen express more AC133 and are more aggressive after in vivo xenograft than cells cultured at 21% oxygen. Thus, oxygen pressure represents a major parameter in the expression of the AC133 molecule but is also important in aggressiveness of glioma cells. The mechanism that links hypoxia, AC133 expression and the tumorigenic behavior of BTSC has been studied. Our results, including CoCl2 induction and shRNA knockdown, suggest a role for HIF-1α in the regulation of AC133 in glioblastoma cells. The second part of our work, immunonanoparticules (ILNC) that target AC133 were synthesized by conjugation of thiolated AC133 or IgG1 isotype control immunoglobulins to lipid nanocapsules (LNC) (4) by linkage through incorporation of lipid PEG(2000) functionalized with reactive-sulfhydryl maleimide groups (DSPE-PEG(2000)-maleimide) on the surface of LNC by using a post-insertion procedure (5). Our results indicate a fixation yield of antibodies on LNC varying from 6 to 60% representing 70 antibodies per LNC. To evaluate the selectivity of immunonanoparticles binding and resulting uptake by cells a second in vitro model has been developed. Human Caco-2 cells, previously described to express AC133, were used and exposed to siRNA targeting the CD133 gene to dispose of two types of Caco-2 cells: Caco-2-AC133high and Caco-2-AC133low. We are now testing ILNC uptake in our Caco-2 cell model. We will test if ILNC are able to bind to AC133 epitope on Caco-2 cells and if they are able to be internalized in cells.

Accumulated results must allow us to extend our knowledge on glioma aggressiveness and to adapt our vectors to resolve the BTSC targeting problem and their death.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A60.
Abstract A70: A genome-wide siRNA screen for modulators of cell death induced by the proteasome inhibitor bortezomib
Authors: Chen Siquan , Blank Jonathan L. , Peters Theodore , Liu Jane , Rappoli David M. , Pickard Michael D. , Menon Saurabh , Yu Jie , Driscoll Denise L. , Lingaraj Trupti , Burkhardt Anne L. , Chen Wei , Sappal Darshan S. , Gray Jesse , Garcia Khristofer , Hales Paul , Leroy Patrick J. , Ringeling John , Rabino Claudia , Spelman James J. , Morgenstern Jay P. , Lightcap Eric S.
Published: June 2012

Multiple pathways have been proposed as the mechanism by which proteasome inhibition induces cell death. To clarify their relative importance, we performed a genome-wide siRNA screen to evaluate the genetic determinants that confer sensitivity of the HCT-116 colon cancer cell line to bortezomib (VELCADE®, PS-341). The screen identified 100 genes whose knock-down affects the lethality of bortezomib. From this list, the accumulation of the proteins ASF1B, Myc, ODC1, PMAIP1 (Noxa), BNIP3, Gadd45α, p-SMC1A, SREBF1, and p53 by proteasome inhibition was linked to the induction of cell death. Fifty-nine genes in the A375 melanoma cell line and 56 genes in the HeLa cervical cancer cell line showed similar interactions with bortezomib to those seen in HCT-116 and a subset of 39 genes were common to all three cell lines. Finally, knockdown of these 100 genes in HCT-116 cells similarly affected their responsiveness to a structurally diverse set of proteasome inhibitors. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A70.
Abstract B1: Antiangiogenic activity of AG-13736, a selective inhibitor of VEGFR-1, -2, -3, in human neuroblastoma
Authors: Rossler Jochen , Farace Francoise , Monet Yann , Opolon Paule , Vassal Gilles , Geoerger Birgit
Published: June 2012

Tumor angiogenesis in neuroblastoma, the most frequent extracranial solid tumor in childhood, has been shown to be an important prognostic factor. High vascularity is reported in aggressive and disseminated neuroblastoma and we previously described all known mechanisms of angiogenesis present, including sprouting of new vessels by angiogenesis growth factors as well as integration of circulating endothelial cells from bone marrow origin. Despite multimodal therapy protocols including surgery, high dose chemotherapy with stem cell support, radiotherapy and differentiating agents, the prognosis of advanced disease remains poor suggesting the evaluation of new therapeutics with distinct mechanism of action like anti-angiogenic agents for neuroblastoma.

We evaluated the new oral pan-VEGFR tyrosine kinase inhibitor AG-13736 in the IGR-N91 xenograft model derived from a primary neuroblastoma bone marrow metastasis. Oral treatment with 30 mg/kg BID during 2 weeks at advanced tumor stage yielded significant tumor growth delay in median time to reach 5 times initial tumor volume of 11.4 days compared to controls (p=0.0006; Mann-Whitney test). AG-13736 treatment down-regulated VEGFR-2 phosphorylation in IGR-N91 and resulted in significantly decreased vessel density and overall surface fraction of tumor vessels in all xenografts as measured by immunohistochemistry using CD34 antibody (mean OSFV at 14 days was 0.56% in treated tumors versus 1.29% in controls; p=0.0006; Mann-Whitney test). We further evaluated the effects of AG-13736 on circulating endothelial cells (CECs) and circulating endothelial progenitor cells (CEPs) measured by flow cytometry. While only a transient reduction was observed in CECs, CEPs were significantly reduced during at least 28 days after treatment (0.40 +/−0.08 cells/ L before treatment compared to 0.02 +/−0.003, 0.00 +/−0.01 and 0.03 +/−0.02 at days 7, 14 and 28, respectively). The mTOR inhibitor rapamycin at 20 mg/kg once weekly resulted similarly to AG-13736 in significant tumor growth delays in IGR-N91 xenografts, however its combination to AG-13736 did not further increase their single agent activities. Thus, AG-13736 is a very potent anti-angiogenic new agent and demands further evaluation in neuroblastoma.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B1.
Abstract B104: The orally bioavailable proteasome inhibitor PR-047 inhibits metastasis in a mouse mammary carcinoma model
Authors: Jiang Jing , Kirk Christopher J. , Muchamuel Tony , Swinarski Debbie , Bennett Mark K.
Published: June 2012

Background: Proteasome inhibitors, such as bortezomib and the tetrapeptide epoxyketone carfilzomib, have shown strong antitumor activity in patients with multiple myeloma and mantle cell lymphoma. Anti-tumor responses are due to direct tumor cell cytotoxicity but may also involve effects on growth factors, angiogenesis and stromal cell interactions. The effect of proteasome inhibitors on metastatic spread has not been well studied in pre-clinical models. PR-047 is a novel orally bioavailable inhibitor of the 20S proteasome, that is structurally related to carfilzomib. PR-047 has demonstrated potent anti-tumor activity in mice bearing subcutaneous tumors of multiple histotypes.

Aim: To evaluate the effect of PR-047 on metastatic spread in mice bearing tumors of the 4T1 mammary carcinoma, an invasive tumor cell line that can spontaneously metastasize from the primary tumor to multiple sites.

Methods: 6–8 week old female BALB/c mice were challenged in the mammary fat pad (metastatic spread model) or via intravenous administration (micro-metastasis model) with 4T1 cells (1 × 105/mouse). Treatment was initiated on Day 3 post tumor challenge in both models. PR-047 was administered orally at 30 or 40 mg/kg on a weekly schedule of QDx2 or QDx5. The cytotoxic agent cyclophosphamide (CY) was used as a positive control. Primary tumors in the mammary fat pad and metastases in lungs were measured.

Results: In the metastatic spread model, PR-047 treatment reduced the number of metastases in the lung by ∼50% without an effect on the growth of the primary tumor. CY reduced the growth of the primary tumor and metastases in the lung. Both dose schedules and dose levels of PR-047 were effective in blocking metastasis. To rule out a direct cytotoxic effect on tumor cells in the lung, we assessed the effect of PR-047 treatment in mice intravenously challenged with 4T1 cells. Unlike CY, which inhibited the growth of micro-metastases, PR-047 treatment had no effect in this model, ruling out a direct cytotoxic effect.

Conclusion: PR-047 blocked the spread of 4T1 tumor cells from the mammary fat pad to the lung without an effect on primary tumor cell growth. This is the first report that an orally bioavailable proteasome inhibitor can block metastasis in vivo. These data support the further evaluation of PR-047 as a novel therapeutic agent for the treatment of metastatic breast cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B104.
Abstract B106: Nedd9 null status initially delays mammary tumor growth, then selects for hyper-aggressive mammary tumors
Authors: Singh Mahendra K. , Izumchenko Eugene , Klein-Szanto Andres JP , Golemis Erica A.
Published: June 2012

Altered expression of the HEF1/CAS-L/NEDD9 scaffolding protein has recently emerged as a candidate mechanism controlling tumor development, progression and metastasis. While some studies have identified elevated NEDD9 expression as pro-metastatic, other works have suggested a negative role in tumor progression. We have recently reported that the Nedd9 null genetic background significantly limits mammary tumor development in the MMTV-polyoma virus middle T (PyVmT) genetic model. Late-developing tumors are characterized by depressed activation of proteins including AKT, SRC, FAK, and ERK, emphasizing an important role of NEDD9 as a scaffolding protein for these pro-oncogenic proteins. However, NEDD9 is also regulates activation of the Aurora-A mitotic kinase (AurA). In vitro, knockdown of NEDD9 reduces AurA activation, suggesting in vivo loss of NEDD9 might be a potential source of genomic instability. We have evaluated a panel of cell lines derived from mammary tumors of MMTV-PyVmT;Nedd9−/− mice. Nedd9−/− cells injected orthotopically in mammary fat pads of SCID mice exhibited significantly faster growth of mammary tumors, and in a tail vein injection assay yielded a dramatically enhanced metastasis to the lungs. As hypothesized, Nedd9−/− cell lines had lower AurA kinase activity than Nedd9+/+ cells, and also had evidence of centrosomal and spindle abnormalities associated with deficient AurA function. Interestingly, the sensitivity of Nedd9−/− cells to small molecule inhibitors targeted at specific partner kinases and to chemotherapeutic agents used in breast cancer notably differed from that of Nedd9+/+ cells. Together, these data support the use of NEDD9 expression as a novel biomarker in establishing prognosis and drug responsiveness of tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B106.
Abstract B107: Targeting metastatic tumor cell functions by Src signaling interference using the small molecule Src inhibitor, saracatinib (AZD0530)
Authors: Siemann Dietmar W. , Dong Meiyu
Published: June 2012

Introduction: The metastatic spread of cancer cells remains the principal cause of treatment failure. Src is a non-receptor tyrosine kinase that is frequently over-expressed in malignances, and increased Src activity may be associated with poor patient prognosis. Src tyrosine kinase plays an important role in tumor cell survival, proliferation, migration and invasion. Since these cellular functions are key components of the metastatic cascade, Src may be a novel target for cancer therapy. The present studies were undertaken to investigate whether molecular intervention in Src associated signaling could reduce the metastatic spread of tumor cells. Specifically, the efficacy of saracatinib a potent, selective and orally available Src inhibitor, was evaluated in the highly metastatic rodent KHT sarcoma cell line.

Methods: The effect of saracatinib treatment on tumor cell functions, including viability, migration, invasion, and attachment, were examined by clonogenicity, migration/invasion and attachment assays, respectively. To assess saracatinib therapeutic effects on metastatic growth, tumor cells were either pretreated with saracatinib prior to intravenous injection or injected intravenously and then the mice were treated with saracatinib. In the latter case three protocols commencing immediately after tumor cell inoculation were investigated: a single 10 mg/kg dose of saracatinib; four 10 mg/kg doses of saracatinib given at 24 hr intervals; daily 10 mg/kg doses of saracatinib administered over a period of 3 weeks; in all protocols saracatinib was administered p.o. Twenty-one days after cell injection the number of lung metastases were determined.

Results: In vitro saracatinib doses ranging from 0.5–5.0 µM significantly reduced Src phosphorylation and functionally inhibited KHT cell proliferation, migration, invasion and adhesion. Pretreating KHT cells with saracatinib (1 or 5 µM, 24 hr) also reduced (2–4-fold) the ability of these cells to form lung metastases when injected into C3H mice via the tail vein. In vivo, daily doses of saracatinib (10 mg/kg) reduced the number of metastases formed at the end of a 3 week period by a factor of ∼2. A similar reduction in lung colonies was achieved with four 10 mg/kg doses of saracatinib given daily commencing immediately after tumor cell injection, or even a single 10 mg/kg treatment administered immediately after tumor cell inoculation.

Conclusions: The present studies explored the possibility of utilizing a molecular targeting strategy directed against key aspects of the metastatic cascade. The present findings demonstrate that saracatinib treatment significantly inhibits the ability of the cancer cells to escape the blood stream and invade at a secondary site supporting the notion that Src signal inhibition may hold considerable promise in impairing metastases formation.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B107.
Abstract B108: Inhibition of osteoblastic tumorigenesis of a novel human prostate cancer cell line by the blockade of TGF-β signaling
Authors: Sun LuZhe , Tang Yuping , Mishra Sweta , De Graffenried Linda , Werner Sherry , Troyer Dean , Thompson Ian
Published: June 2012

The skeleton is the most common site of prostate cancer metastasis, which often results in osteoblastic lesions. The associated fractures and pain are major causes of morbidity and mortality in prostate cancer patients. Transforming growth factor-beta (TGFβ) signaling has been shown to developmentally regulate bone mass and bone matrix properties. It has also been shown to promote osteolytic metastasis induced by breast cancer through its regulation of some secreted proteins that are involved in bone resorption. However, its role in prostate cancer-induced osteoblastic metastasis is much less clear. This is in part due to a lack of prostate cancer cell lines that can efficiently induce bone metastasis and osteoblastic lesions in animal models. We found that a newly established human prostate cancer PacMetUT1 cell line produces copious amounts of active TGFβ1 and induces extensive bone metastases in calvaria, rib, femur, and tibia when inoculated in the left cardiac ventricle of male nude mice. Histology revealed extensive bone formation in the metastatic lesions of the calvaria and femur with some areas showing bone resorption. Stable knockdown of TGFβ1 with a shRNA in the PacMetUT1 cells resulted in significantly reduced TGFβ1 secretion and decreased tumor incidence when the cells were directly injected into the tibiae of male nude mice. Systemic administration of either a small inhibitor of TGFβ type I receptor kinase or a pan TGFβ binding protein also decreased osteoblastic intratibial tumor incidence and growth. PacMetUT1 is a novel prostate cancer cell line for the investigation of mechanisms that regulate formation of metastatic osteoblastic bone lesions. Future studies will reveal whether targeting the TGFβ signaling pathway is feasible for the prevention and treatment of prostate cancer-induced bone metastasis.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B108.