bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C207: Checkpoint abrogation and potentiation of cytotoxic chemotherapeutics with a novel checkpoint kinase 1 inhibitor
Authors: Massey Andrew J. , Stokes Stephen , Browne Helen , Borgognoni Jennifer , Foloppe Nicolas , Scrace Simon , Fiumana Andrea , Bedford Simon , Webb Paul , Ray Stuart , Jordan Allan , Drysdale Martin J. , Wood Mike
Published: June 2012

Conventional chemotherapeutic agents such as gemcitabine, cisplatin or irinotecan induce DNA damage and activate cell cycle checkpoints. P53 defective tumors lack a functional G1 checkpoint and rely heavily on the S and G2 checkpoints, and the effector kinase Chk1, for protection against this DNA damage. Inhibiting Chk1 potentiates the anti-tumor effects of these cytotoxic chemotherapeutic agents. Targeting Chk1 is a potential therapeutic opportunity for potentiating the anti-tumor efficacy of DNA damaging cytotoxic chemotherapeutic drugs without increasing their toxicity to normal cells. Elaboration of a designed kinase directed fragment core utilizing X-ray structure guided design lead to the identification of a potent pyridone series of Chk1 inhibitors. Further profiling identified V158411 as the lead candidate. X-ray crystallography identified V158411 as being bound to the ATPase site in the kinase domain of Chk1. V158411 potently inhibited Chk1 and Chk2 with IC50s of 4.4 and 4.5nM respectively. The addition of V158411 to gemcitabine or camptothecin treated cells abrogated the cell cycle checkpoints induced by these agents resulting in the expected modulation of cell cycle proteins and increased apoptosis. V158411 potentiated the cytotoxicity of gemcitabine, cisplatin, SN38 and camptothecin in a variety of p53 deficient but not proficient human tumor cell lines in vitro. V158411 could be formulated in a simple aqueous form suitable for i.v. dosing. In nude mice, V158411 was well tolerated as a single agent (MTD >100mg/kg) and in combination with irinotecan. Intravenous administration to rats and mice resulted in low plasma clearances (20mL/min/kg) and long half-lives (2.9–3.7h). In tumor bearing animals, V158411 was detected at high concentrations in the tumor (tumor:plasma AUC ratio of 4.7) with a long tumor elimination half life of 22 hours. No pharmacologically relevant in vivo drug-drug interaction with irinotecan was identified. V158411 potentiated the anti-tumor activity of gemcitabine and irinotecan in a variety of human tumor xenograft models without additional systemic toxicity. These results demonstrate the potential of combining V158411 with standard of care chemotherapeutic agents to potentiate the therapeutic efficacy of these agents without increasing their toxicity to normal cells. Based on this data, the clinical development of V158411 is currently being actively pursued.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C207.
Abstract C212: Pharmacological and pharmacokinetc characterization of RP1040, a novel and potent c-Met tyrosine kinase inhibitor
Authors: Viswanadha Srikant , Muthuppalaniappan Meyyappan , Varanasi Kanthikiran VS , Veeraraghavan Sridhar , Thainashmuthu Josephrajan , Murugan Sivan Perumal , Kakarla Prasad Babu , Kuppireddi Sumalatha , Vakkalanka Swaroop
Published: June 2012

Background: Receptor tyrosine kinases (RTK) represent a class of high affinity cell surface receptors that play a critical role in the development and progression of several types of cancers. Among the receptor tyrosine kinases, c-Met mediates diverse cellular responses critical for tumor progression, growth, and survival. Alteration of the c-Met signalling cascade represents an attractive approach aimed at blocking invasion and metastasis of cancer cells. The objective of this study was to investigate the pharmacological and pharmacokinetic properties of RP1040, a novel small-molecule inhibitor of c-Met kinase.

Methods: c-Met Kinase activity of RP1040 was determined using using an HTScan® recombinant human Met Kinase Assay Kit (Cell Signaling Technology, Beverly, MA) with modifications. Viability assay (MTT) was conducted to determine the growth inhibitory effect of the compound on the high c-Met expressing sk-LMS-1 cell line. Metabolic stability of the compound was evaluated in microsomes obtained from mouse, rat, dog, monkey, and human. Pharmacokinetic behaviour of RP1040 in plasma after single dose oral administration or IV injection was determined in male Wistar rats.

Results: RP1040 demonstrated remarkable potency against the purified Met kinase by inhibiting enzyme activity at low nanomolar concentrations (IC50 = 1.3 nM). In addition, the compound caused a significant reduction in viability of sk-LMS-1 cells stimulated with 100 ng/ml HGF (IC50 = 0.03 nM). Pharmacokinetic studies indicated good oral absorption of RP1040 with plasma concentrations above 100 nM up to 24 h post administration. The compound had an elimination half-life of 8.95 hr with a clearance value of 4.21 ml/min/kg indicating a propensity to act as a long acting drug in this class. Further, RP1040 was metabolically stable across the species studied.

Conclusions: Our results demonstrate that RP1040 is a potent c-Met kinase inhibitor with a favourable pharmacokinetic profile and superior IC50 values compared to existing c-Met kinase inhibitors in development. RP1040 is currently being tested for in vitro and in vivo efficacy across various cancer cell lines and xenograft models besides selectivity against other receptor tyrosine kinases.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C212.
Abstract C213: Sorafenib blocks tumor growth, angiogenesis, and metastatic potential in preclinical models of osteosarcoma through the inhibition of ERK1/2, MCL-1, and ezrin pathways
Authors: Pignochino Ymera , Grignani Giovanni , Cavalloni Giuliana , Tapparo Marta , Motta Manuela , Bruno Stefania , Bottos Alessia , Gammaitoni Loretta , Camussi Giovanni , Migliardi Giorgia , Alberghini Marco , Torchio Bruno , Ferrari Stefano , Picci Piero , Fagioli Franca , Aglietta Massimo
Published: June 2012

Osteosarcoma (OS) is the most common primary bone tumor in children and young adults. Despite improved prognosis, metastatic or relapsed OS remains largely incurable and no significant improvement has been observed in the last 20 years. Therefore, the search for alternative agents in OS is mandatory.

We explored phospho-ERK 1/2, MCL-1, phospho-Ezrin/Radixin/Moesin (P-ERM) as potential therapeutic targets. The activation of these pathways was shown by immunohistochemistry in about 70% of cases and in all OS cell lines analyzed. Mutational analysis revealed no activating mutations in KRAS. While, BRAF gene was found mutated in 4/30 OS samples from patients. Based on these results we tested the multikinases inhibitor sorafenib (BAY 43-9006) in preclinical models of OS. Sorafenib inhibited OS cell lines proliferation, induced apoptosis and downregulated P-ERK1/2, MCL-1, P-ERM in a dose dependent manner. ERM dephosphorylation is not due to ERK inhibition. The downregulation of MCL-1 increased apoptosis in OS cell lines. In chick embryo chorioallantoic membrane the angiogenesis induced by OS supernatants was blocked by sorafenib treatments. Indeed, sorafenib reduced VEGF and MMP2 production. Sorafenib treatment dramatically reduced tumor volume of OS xenografts and lung metastasis in SCID mice.

In conclusion, ERK1/2, MCL-1 and ERM pathways are active in OS. Sorafenib is able to interrupt their routes in vitro and in vivo displaying antitumoral activity, antiangiogenic effect and reducing metastasis colonies formation in lungs. These data support the testing of sorafenib as a potential therapeutic option in metastatic or relapsed OS patients failing standard treatments.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C213.
Abstract C227: Doxorubicin induces AKT phosphorylation through activation of the aryl hydrocarbon receptor in the heart
Authors: Maria Volkova , Ozcan Cevher , Palmeri Monica , Russell Raymond
Published: June 2012

Doxorubicin (DOX) is one of the most effective anticancer drugs in treating different types of tumors. However, cumulative cardiotoxicity is a significant side effect of this therapy resulting in clinical heart failure through the production of reactive oxygen species (ROS). However, the protective responses of the heart to DOX treatment are still unknown. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is primarily involved in the detoxification of xenobiotic agents that generate ROS. Activation of AhR results in its migration to the nucleus where it dimerizes with ARNT1 to form a transcription factor for genes coding many phase I drug metabolizing enzymes, such as the cytochrome P450, CYP1A1. We hypothesize that DOX treatment activates AhR and triggers Akt phosphorylation as a protective mechanism against oxidant stress. H9C2 cells incubated with 2.5 M DOX for 4 hours showed a significant increase of AhR protein levels in the nuclear fraction, which then decreased to baseline levels by 24 hours of treatment. The same pattern of change was seen for CYP1A1 expression. Immunoprecipitation of the nuclear protein fraction with AhR antibodies and subsequent immunoblotting with ARNT1 antibodies showed increased binding between these proteins after 4 hours of DOX treatment. Similar results were shown when ARNT1 immunoprecipitation and AhR immunoblotting were performed. Increased phosphorylation of Akt at Ser473 was detected at 4 hours of DOX treatment, which decreased by 24 hours. A similar temporal pattern of phosphorylation was identified for known downstream targets of Akt (pSer9-GSK3 and pSer136-BAD). Transfection with either AhR or ARNT1 siRNAs blocked the phosphorylation of Akt following DOX treatment in H9C2 cells, increased ROS production as well as cytotoxicity. DOX treatment of isolated adult rat cardiomyocytes showed similar pattern of changes in Akt phosphorylation and its downstream targets as well as increased oxidative stress. In mice injected with 10 mg/kg DOX, AhR translocated into the nucleus by 4 hours after injection, which was associated with ARNT1 binding and increased CYP1A1 expression. Phosphorylation of Akt, GSK3 and BAD increased by 24 hours after DOX injection. Oxidative stress was significantly higher in mice injected with DOX after 4 hours. We conclude that the early cardiac response to DOX treatment involves Akt phosphorylation through AhR activation.Moreover, silencing of AhR might lead to higher oxidative stress and increased cellular toxicity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C227.
Abstract C231: A great mime produced by nature: The case of paclitaxel
Authors: Ferlini Cristiano , Raspaglio Giuseppina , Cicchillitti Lucia , Bartollino Silvia , Gallo Daniela , Persico Marco , Fattorusso Caterina , Scambia Giovanni
Published: June 2012

Paclitaxel is widely used in the medical treatment of solid tumors. Despite the clinical success still now the exact mechanism(s) through which the drug kills cancer cells are unknown. In this work we provide evidence, through Surface Plasmon Resonance technology, that paclitaxel beside to microtubules is able also to target mitochondria by directly interacting with the disordered loop of Bcl-2, thereby stimulating the opening of the permeability transition pore channel and ultimately reverting Bcl-2 function from antiapoptotic to proapoptotic. Studying the paclitaxel binding site in Bcl-2, we discovered through molecular modeling that it is positioned in the disordered loop domain and is extraordinarily similar to that experimentally defined in beta-tubulin, thereby prompting us to speculate that paclitaxel is a peptidomimetic factor. Since to our knowledge, the only factor able to revert the Bcl-2 function from protector to cell killer is Nur77, we tested the hypothesis that paclitaxel mimics the activity of Nur77. To address this issue, coimmunoprecipitation experiments demonstrated that like paclitaxel Nur77 interacts with Bcl-2 in the disordered loop domain and with beta-tubulin. Moreover, stably transformed cells with Nur77 show the classical slow band isoform of Bcl-2 detectable in cells treated with paclitaxel. Finally, interaction between Nur-77 and Bcl-2 was inhibited by paclitaxel, thus demonstrating that the drug and Nur77 compete for the binding to Bcl-2. The discovery of the molecular component mimicked by paclitaxel could promote the development of novel agonists of the paclitaxel receptor with a nontaxane structure.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C231.
Abstract C232: ALB-109564, a novel tubulin inhibitor with improved efficacy over vinorelbine, is better tolerated when dosed iv versus ip, leading to improved activity in human tumor xenograft studies
Authors: Wolf Mark A. , Johnson Randall K. , Xie Deijian , Avrutskaya Anna , Mullin Robert , Godfrey Beverly , Mead Mary Ann , Trachet Erin E. , Leopold Wilbur R. , Guzzo Peter , Scott Ian
Published: June 2012

Purpose: ALB-109564 is a novel semi-synthetic Vinca alkaloid which is currently in a Phase I clinical trial. The antitumor activity of ALB-109564 was compared to vinorelbine in human tumor xenograft studies when dosed ip and iv.

Methods: The maximum tolerated dose (MTD) of ALB-109564 and vinorelbine was determined in athymic nude mice using both ip and iv dosing on q4dx4 and q7dx4 schedules. Athymic nude mice (n = 10) were implanted sc with a number of human tumor cell lines. Following establishment of measurable tumors, mice received equivalent therapeutic doses, based on their MTDs, of vinorelbine and ALB-109564. Vinorelbine and ALB-109564 were compared by dosing ip (q4dx4) in a panel of five xenografts (H460 NSCLC, Colo205 colon cancer, PC3 prostate cancer, H69 SCLC, and MX-1 breast cancer). Additionally, a comparison of ALB-109564 and vinorelbine was run dosing iv on a q7dx4 schedule in two xenograft models (PC3 and H460).

Results: It was discovered that ALB-109564 and vinorelbine were better tolerated when dosed iv rather than ip, allowing higher dose levels of both agents. For example, the MTD for ALB-109564 using the q4dx4 schedule was 6 mpk ip compared with 14 mpk iv. ALB-109564 demonstrated antitumor activity superior to vinorelbine when dosed at their respective MTDs ip. Statistically significant tumor growth delays (TGDs) were observed with ALB-109564 in four of the five xenografts studied while vinorelbine did not significantly delay tumor growth in any of the models when dosing ip. A comparison of the two agents dosed iv in the PC3 and H460 xenograft models showed that both agents induced significant TGDs in both models with ALB-109564 showing superior efficacy in the H460 study (35.7 days TGD vs. 15.7 days at respective MTDs).

Conclusions: Preclinical evaluation of tubulin inhibitor ALB-109564 (12′-methylthiovinblastine dihydrochloride) demonstrated in vivo oncolytic activity against several human tumor cell types in xenograft models greater than that seen with vinorelbine. These results demonstrate that minor structural modifications of vinca alkaloids can have a large impact on activity. Based on these results a Phase I clinical study with ALB-109564 was initiated in August 2008 and is ongoing through dose-escalation.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C232.
Abstract C235: OXi4503: A novel vascular disrupting agent that enhances radiation-induced local tumor control when given alone or as part of a trimodality therapy
Authors: Horsman Michael R. , Nomura Miwako , Hokland Steffen
Published: June 2012

Background: OXi4503 is a novel vascular disrupting agent (VDA) with single agent activity against solid tumors. However, its potential application in cancer therapy lies in its combination with more conventional therapies. The aim of this study was to determine its interaction with radiation when given alone or as part of a trimodality treatment schedule that also included cisplatin or mild temperature hyperthermia.

Material and Methods: Restrained non-anaesthetised CDF1 mice, with a 200 cubic mm C3H mammary carcinoma grown in the right rear foot, were used. Tumors were locally irradiated (240 kV x-rays) with single graded radiation doses. OXi4503 (50 mg/kg) and cisplatin (6 mg/kg) were prepared fresh before each experiment and injected intraperitoneally in a saline mixture at 0.02 ml/g body weight. Previous tumor growth studies had shown that the combination of OXi4503 with radiation was independent of timing and sequencing, thus it was always given within the first 60 minutes after irradiating. But, for cisplatin the optimal effect with radiation and/or OXi4503 was when the cisplatin was administered 3-hours later. Mild temperature hyperthermia (41.5°C; 1-hour) involved immersing the tumor bearing foot in a water bath and was always started 4-hours after irradiating. The response endpoint was the percentage of mice in each treatment group showing local tumor control 90 days after irradiating. From full radiation dose response curves, the TCD50 values (radiation dose to control 50% of tumors) were estimated. A Chi-squared test (significance level of p<0.05) was used to statistically compare the TCD50 results.

Results: The TCD50 value (with 95% confidence interval) for radiation alone was 53 Gy (50–56). Irradiating tumors and then injecting OXi4503 significantly reduced this to 43 Gy (40–44). Injecting cisplatin 3-hours after irradiating had absolutely no effect on radiation response; the TCD50 being 53 Gy (50–56), but combining all 3 modalities gave the lowest TCD50 value of 41 Gy (38–43). Mild temperature hyperthermia produced a small yet significant reduction in the radiation TCD50 value to 47 Gy (45–50), But, the greatest enhancement was obtained when radiation, OXi4503 and heat were combined. With this combination the TCD50 was significantly reduced to 37 Gy (34–41).

Conclusions: OXi4503 on its own significantly enhanced the response of this C3H mammary carcinoma to radiation. Although cisplatin and mild hyperthermia had little or no effect on radiation-induced local tumor control, combining these therapies with OXi4503 resulted in the greatest enhancement of radiation response. This VDA clearly has a role to play in combination with radiation therapy whether used on its own or as part of a multimodality schedule.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C235.
Abstract C236: A novel oxime-containing flavone anticancer agent, WTC01, exhibits potent microtubule disruption ability
Authors: Juang Shin-Hun , Lee Chao-Hsun , Lin Yu-Chin , Chaing Chang-Ying , Wang Tai-Chi
Published: June 2012

Flavonoids and its derivatives are common components found in human diet. Several studies indicated flavonoids could benefit in cancer prevention or therapy, but their structure-activity relationships (SAR) remain poorly defined. In order to further understand the SAR, a series of oxime-containing flavone derivatives were designed, synthesized and evaluated for their biological anti-cancer potential. The anti-proliferative ability of these compounds was measured by several human cancer cell lines from different origins including nasopharyngeal cancer (NPC-TW01), leukemia (MT-2) and lung carcinoma (H661). Among over 30 different oxime-containing flavone derivatives, (Z)-6-[2-hydroxyimino-2-(4-methoxyphenyl)ethoxy]-2-phenyl -4H-1- benzopyran-4-one (WTC01) possessed potent activity against NPC-TW01 growth with IC50 of 400 nM. As determined by flow cytometry, WTC01 treatment results in an accumulation of NPC-TW01 cells in G2-M phase following a 6-h exposure with amaximum accumulation observed by 18 h. Furthermore, Annexin-V/propidium iodide (PI) binding assay, Hoechst 33258/PI double staining, and PARP cleavage indicates that cell death proceeded through an apoptotic pathway. Interestingly,WTC01 also causes mitochondrial transmembrane potential loss and activation of caspase-9 and caspase-3 without noticeable activation of the caspase-8. These results suggest that the WTC01-mediated apoptotic signaling pathway depends on the mitochondria and caspase-9/-3 cascades. Further studies showed that WTC01 inhibits microtubule assembly in a concentration-dependent manner, this data suggests that tubulin may serve as the cellular target for WTC01. Taken together, these findings indicated that WTC01, a novel synthetic oxime-containing flavone, exhibits potent activity against cancer growth through the disruption of microtubule and has potential for management of nasopharyngeal malignancies.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C236.
Abstract C24: Methylation of AKAP12 promoter in lung cancer
Authors: Kim Yeul Hong , Jo Uk Hyun , Whang Young Mi , Sung Jae Sook , Park Kyong Hwa
Published: June 2012

AKAP12 play an important role in tumor growth suppression by inducing apoptosis with the regulation of multiple molecules in the cell cycle progression. This function of AKAP12 gene might be inhibited by epigenetic repression like other tumor suppressor genes. In the previous report, we showed that AKAP12 inactivation was associated with the promoter methylation in the lung cancer. In the present study, to investigate whether the promoter methylation of AKAP12 gene affects the expression of AKAP12 protein, we carried out in vitro methylation assay and protein analysis. We also performed methylation profiling of AKAP12 promoter using the bisulfite-sequencing in the additional matched normal and lung cancer tissue DNA, and serum DNA samples. The expression of the AKAP12 protein was down-regulated in lung cancer cells. The reduced expression of AKAP12 protein was restored by the treatment of the DNA methyltransferase inhibitor. AKAP12 fragments pGL3-837 M (methylated) and pGL3-443 M (methylated) showed low luciferase activities compared to the unmethylated constructs. CpG islands of the AKAP12 promoter were frequently methylated in tumor samples compared with normal tissue samples. Furthermore, CpG islands methylation of AKAP12 was more frequently found in non-relapse patients after surgery compared to early relapse patients within 2 years after surgery.

In conclusion, our results demonstrated that AKAP12 expression was regulated by DNA methylation and AKAP12 promoter methylation was associated with lung cancer development and prognosis after surgery. This work was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A010250) and Seoul Research and Business Development Program (10574)

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C24.
Abstract C247: Noninvasive optical imaging of the tumor microenvironment within the hollow fibre assay: A model for the evaluation of novel anticancer agents
Authors: Whelband Elisabeth , Kumari Rajendra , Grabowska Anna M. , Argent Richard H. , Shams-Nateri Abdol , Watson Susan A.
Published: June 2012

The hollow fibre assay (HFA) facilitates the short-term assessment of cancer drug efficacy in vivo, bridging in vitro screening methods and more complex xenograft in vivo models. However a drawback of this model is the need for end-stage cell recovery and assessment in order to determine the test compound effects. An additional limitation of this system is the use of a homogenous population of epithelial cells, which is not representative of the solid tumor micro-environment, a critical component in tumor progression and the development of drug resistance. The aims of our current research were to recapitulate the human tumor micro-environment within the hollow fibre model and apply a real-time bio-imaging component to enable the non-invasive analysis of drug responsiveness and toxicity in addition to evaluate markers of tumor progression, which was confirmed histologically.

Using epithelial tumor cells stably expressing fluorescent/bioluminescent reporter genes, we have investigated cell viability and the response to cytotoxic treatment in the presence and absence of human mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) both within in vitro cell cultures and the HFA. Real-time imaging of bioluminescent tumor cells demonstrated that these cells can actively proliferate for up to 16 days within the hollow fibres. In the presence of MSCs or CAFs tumor growth was significantly enhanced compared with tumor cells alone and immuno—staining analysis revealed stromal deposits around the periphery of the fibre with MSCs acquiring a ‘myofibroblastic’ phenotype with prolonged culture, resulting in growth promoting effects similar to CAFs. Tumor cells expressing dual reporters with luciferase under the control of 3 hypoxia response elements (HREs) and RFP driven by a CMV promoter confirmed the development of hypoxia within the HFA and enabled further investigation of cancer stem cell development and epithelial:mesenchymal transition. This re-capitulation of the human tumor micro-environment provides a relevant model by which to identify and characterise novel anti-cancer compounds.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C247.
Abstract C249: Down-regulation of the HIF pathway enhances anti-tumor effects of the mTOR inhibitor ridaforolimus in the Her2/Neu mouse model of breast cancer
Authors: Di Bacco Alessandra , C. Artime Marlene , Gargano Diana , Menon Mary-Kamala , Rao Sudhir , Zhang Weisheng , Haines Brian B. , Marszalek Joseph
Published: June 2012

The Hypoxia-inducible Factor (HIF) pathway is a unique target in cancer because it controls multiple processes involved in tumor progression including neovascularization, tumor cell metabolism, invasion and metastasis. Hypoxia is elevated in a variety of tumor types and associated with resistance to therapeutics resulting in poor prognoses for patients. While a variety of tumor xenograft tumor models have demonstrated sensitivity to HIF pathway inhibition, studies in transgenic mouse models may provide a better understanding of the response as it more closely resembles the disease. Here we evaluate the role of HIF pathway in the MMTV-Neu transgenic (NeuT) breast cancer model and influence of the HIF pathway on response to the mTOR inhibitor ridaforolimus/MK-8669. A HIF regulated neuT model was generated by crossing NeuT transgenic mouse with one expressing a doxycyline-inducible HIF1β shRNA (NeuT-HIF1β) (Taconic-Artemis, Cologne, Germany). HIF1β is the obligate heterodimerization partner for the HIFα subunits and is required for HIF pathway activity. Upon derepression of the HIF1β shRNA by doxycycline treatment, greater than 80% knockdown is observed in most tissues. After 21-days of shRNA induction, tumor growth suppression was observed in the doxycyline-treated NeuT-HIF1β mice, but not NeuT littermate control mice. Growing evidence suggests that in hypoxia mTOR activity is often inhibited by HIF dependent and independent mechanisms. To evaluate the effect of hypoxia on the sensitivity of cells to a mTOR inhibitor, four cell lines (C786; U251; H1915; SKLU1) were treated with ridaforolimus cultured under normoxic (21% oxygen) and hypoxic (0.1% oxygen) conditions, the later oxygen concentration observed in hypoxic region of tumors. Under normoxia, the growth rate for all four cell lines was reduced by ridaforolimus. Under hypoxia, two lines were unaffected (786; U251) by ridaforolimus, while the other (H1915; SKLU1) remained sensitive. Histological analysis of the neuT tumors revealed that PI3K/mTOR and HIF pathways activities are mutually exclusive in these tumors. PI3K/mTOR pathway activity was detected in normoxic regions and HIF pathway activity present only in hypoxic regions. Thus we hypothesized targeting both pathways should achieve a far greater effect than each treatment separately. Treatment of mice with HIF1β knockdown or ridaforolimus resulted in 2-fold reduction in tumor growth and the combination resulted in a 4-fold decrease when both two pathways were targeted together. Histological analysis of these tumors will be discussed. In conclusion these data demonstrate that the effect of ridaforolimus on tumor growth can be improved by combining treatment that reduces the level of HIF pathway activity in tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C249.
Abstract C32: First-in-human clinical, pharmacokinetic (PK) and pharmacodynamic (PD) phase I study of the first-in-class spliceosome inhibitor E7107 administered IV (bolus) on days 1, 8, and 15 every 28 days to patients with solid tumors
Authors: Eskens Ferry A. , Ramos Francisco J. , Burger Herman , de Jonge Maja JA , Wanders Jantien , Lopez-Anaya Arturo , Baselga Jose , Tabernero Josep
Published: June 2012

Background: E7107 is a first-in-class inhibitor of the spliceosome, most likely by interacting with spliceosome-associated protein-130 (SAP 130). Splicing removes intron sequences from pre-mRNA and exons are fused resulting in the formation of mature mRNA. Alternative splicing frequently encodes oncoproteins. E7107 interferes the maturation process of pre-mRNA to mRNA, with consequent changes in protein expression profiles.

Methods: Objectives of this study were to explore (1) tolerability and safety profile, (2) PK, (3) PD effects on pre-mRNA splicing, and (4) anti tumor activity of E7107 administered as bolus infusion on days 1, 8, 15 of a 28-day schedule

Results: 36 patients (21M/15F, median age 61yrs (45–79)) received E7107 doses of 0.6 mg/m2 (n=4), 0.9 mg/m2 (n=3), 1.3 mg/m2 (n=3), 2.0 mg/m2 (n=3), 3.0 mg/m2 (n=4), 4.5 mg/m2 (n=3) and 4.0 mg/m2 (n=16). At 4.5 mg/m2 two episodes of DLT (grade 3 and 4 diarrhea) and at 4.0 mg/m2 one episode of DLT (a combination of grade 3 nausea, vomiting and abdominal cramps) were observed. Other frequently occurring side effects were mainly gastrointestinal. After drug discontinuation, one patient at 4.0 mg/m2 experienced reversible grade 4 blurred vision. The maximum tolerable dose (MTD) is 4.0 mg/m2. No complete or partial responses were observed. Pharmacokinetic analysis revealed large volume of distribution (Vss: 279 to 1369 L), high systemic clearance (CL: 111 to 253 L/hr), and moderate elimination half-life (t1/2: 5.3 to 15.1 hr). Systemic exposure on days 1 and 15 (Cmax, AUC0-∞) increased in a dose-dependent manner. At the MTD, mRNA levels of selected target genes (TRAPPC4, SLC25A19, GTF2H1), monitored in PBMC's, showed a 15–25-fold decrease, whereas unspliced pre-mRNA levels of DNAJB1 and EIF4A1 showed a 10–25-fold increase. Notably, at days 1 and 15, modulations generally peaked at 2–6 hr after end of the infusion and almost completely recovered to base-line levels at 24–48 hr.

Conclusion: The MTD for E7107 using this schedule is 4.0 mg/m2. PK is dose-dependent and reproducible within subjects. PD analysis revealed dose-dependent reversible inhibition of pre-mRNA processing of target genes, confirming proof-of-principle activity of E7107.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C32.
Abstract C34: p53 overexpression is correlated with poor outcome in premenopausal women with breast cancer treated with tamoxifen after chemotherapy
Authors: Yom Cha Kyong , Kim Hee Jeong , Kim Jun Ho , Ahn Sei Hyun , Park Yong Lai , Kim Hee Sung
Published: June 2012

Purpose: The aims of this study were to evaluate the difference in outcomes based on p53 overexpression of breast cancer patients who received adjuvant therapy following local treatment for invasive ductal carcinoma, not otherwise specified (NOS).

Patients and Methods: We analyzed data from 4,683 cancer patients enrolled in 2 institutions between 1997 and 2006. We analyzed the correlation between p53 overexpression and relapse, response to adjuvant therapy, breast cancer-specific survival (BCSS), and relapse-free survival (RFS) in patients with primary breast cancer.

Results: p53 overexpression was noted in 1,091 patients (23.3%). A significant correlation existed between p53 overexpression and poor prognostic factors, an increased frequency of regional recurrence, visceral metastasis, and worse BCSS and RFS. Based upon subgroup analyses, combined age (< 35 years, 35–50 years, and > 50 years) and adjuvant therapy (hormone therapy only, chemotherapy only, and hormone therapy following chemotherapy), the greatest reduction of survival based on p53 overexpression was noted in patients 35–50 years of age who received hormone therapy following chemotherapy (p<0.05). Multivariate analysis showed that p53 overexpression is an independent prognostic factor in patients treated with hormone therapy and chemotherapy (relative risk for BCSS, 2.003; 95% CI, 1.105–3.631; p=0.022).

Conclusion: p53-overexpressing breast cancer patients between 35 and 50 years of age who received tamoxifen following chemotherapy had the greatest adverse effect on outcome. p53 overexpression is significantly associated with tamoxifen-resistance in premenopausal women with breast cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C34.
Abstract C62: Identification of GSK2126458, a highly potent inhibitor of phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR)
Authors: Knight Steven D. , Adams Nicholas D. , Burgess Joelle L. , Chaudhari Amita M. , Darcy Michael G. , Donatelli Carla A. , Newlander Ken A. , Parrish Cynthia A. , Ridgers Lance H. , Sarpong Martha A. , Schmidt Stanley J. , Van Aller Glenn , Carson Jeff D. , Elkins Patricia A. , Diamond Melody , Gardiner Christine M. , Garver Eric , Luo Lusong , Raha Kaushik , Sung Chiu-Mei , Tummino Peter J. , Auger Kurt R. , Dhanak Dashyant
Published: June 2012

Phosphoinositide 3-kinase (PI3K) is a critical regulator of cell growth and transformation and its signaling pathway is one of the most commonly mutated pathways in human cancer. The mammalian target of rapamycin (mTOR), a class IV PI3K protein kinase, is also a central regulator of cell growth, and mTOR inhibitors are believed to augment the antiproliferative efficacy of the PI3K/AKT pathway. GSK1059615, our first PI3K clinical compound, progressed to a dose escalation study in patients with refractory malignancies. Following the discovery of GSK1059615, we sought to identify a second inhibitor with improved potency, selectivity, and pharmacokinetics. Key to our approach to achieving the desired levels of PI3K activity was to pursue structure-based design utilizing crystallography of the more amenable PI3K as a surrogate protein. Following a chemistry lead optimization effort, the pyridylsulfonamide GSK2126458 was identified as a highly potent, orally bioavailable, pan-PI3K and mTOR inhibitor (PI3K app Ki = 19 pM; mTORC1 app Ki = 180 pM; mTORC2 app Ki = 300 pM). Consistent with potent PI3K and mTORC2 enzyme inhibition, GSK2126458 decreased cellular levels of phosphorylated AKT (BT474 pAKT IC50 = 180 pM) and inhibited cell proliferation in a large panel of cancer cell lines (e.g. BT474 growth IC50 = 2 nM). GSK2126458 showed good exposure in four pre-clinical animal species and exhibited in vivo activity in both pharmacodynamic and tumor growth efficacy models. GSK2126458 is being evaluated currently in human clinical trials for the treatment of cancer. The discovery, design, and optimization of GSK2126458 and related analogs will be presented.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C62.
Abstract C68: Endogenous expression of an oncogenic PI3K mutation leads to epithelial-mesenchymal transition (EMT) and invasiveness
Authors: Wallin Jeffrey J. , Guan Jane , Haverty Pete , Modrusan Zora , Arena Sabrina , Yao Evelyn , Hoeflich Klaus P. , Bardelli Alberto , Torrance Christopher , Belvin Marcia , Friedman Lori S.
Published: June 2012

The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents a therapeutic target. In order to investigate the primary phenotypes mediated by mutant PIK3CA in a controlled and patient-relevant context, we utilized human non-tumorigenic MCF10A parental and isogenic knock-in cell lines that harbor a common activating PIK3CA mutation (H1047R). We found that endogenous introduction of a mutated PIK3CA allele results in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype compared to isogenic wild-type cells. Moreover, a potent and selective inhibitor of PIK3CA (GDC-0941) was highly effective in reversing this phenotype in 3D cell culture. Our results suggest that invasion assays and biomarkers should be used to reveal PIK3CA ‘oncogeneaddiction’ responses of pathway-targeted agents.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C68.
Abstract C70: Targeting tumor cell glycolysis: Discovery and characterization of small molecule inhibitors of IPFK2 (Inducible 6-Phosphofructo-2-Kinase)
Authors: Markby David W.
Published: June 2012

Background: Cancer cells frequently show high rates of glycolysis even under normoxic conditions (the Warburg effect). Cellular glycolytic flux is regulated in part at the level of 6-phosphofructo-1-kinase (PFK1) by a potent allosteric activator, fructose-2,6-bisphosphate (F2,6BP), whose concentration is controlled by a family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1–4). Among the isozymes, PFKFB3 (or inducible 6-phosphofructo-2-kinase, IPFK2) exhibits the highest ratio of kinase to phosphatase activity and has been implicated in cancer progression by tumor overexpression and siRNA interference studies. Hence, inhibition of IPFK2 activity may selectively target tumor metabolism by blocking glycolysis and cell proliferation.

Methods: Small molecule inhibitors of IPFK2 were identified by high-throughput screening and lead optimization using in vitro enzymatic assays and cellular assays for F2,6BP. Glycolytic pathway activity was monitored by lactate production, extracellular acidification rate (ECAR), and cellular F1,6BP level. Mitochondrial function was assessed by measuring oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP). Cellular ATP level was used to assess short term effects on bioenergetics and long term effects on viability. PFKFB activity in vivo in tumor xenografts and normal tissue was measured by F2,6BP level.

Conclusions: IPFK2 inhibitors displayed potent biochemical activity (IC50 1–10 nM) and were competitive with ATP and noncompetitive against fructose-6-phosphate. Treatment of cells with IPFK2 inhibitors reduced cellular F2,6BP levels with IC50 values 50–500 nM under both normoxia and hypoxia, consistent with specific knockdown of IPFK2 in siRNA experiments. IPFK2 inhibitors reduced glycolytic flux as assessed by ECAR and cellular F1,6BP levels, although the extent of inhibition varied widely among cell lines. Short term treatment with these compounds generally did not inhibit mitochondrial function (as assessed by OCR and MMP) or reduce cellular ATP levels. Longer term treatment with IPFK2 inhibitors reduced tumor cell viability under low serum conditions. In vivo pharmacodynamic studies with orally bioavailable compounds demonstrated target inhibition in mouse xenograft tumor models as assessed by a reduction in F2,6BP. Further lead optimization and biological characterization is underway with the goal of identifying an IPFK2-selective glycolytic inhibitor for development as a cancer therapeutic.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C70.
Abstract C8: Short-chain fatty acids induce autophagy through inhibition of mTOR pathway
Authors: Tang Yong , Chen Yakun , Jiang Hongmei , Nie Daotai
Published: June 2012

Short-chain fatty acid (SCFA), mainly propionate and butyrate are major by-products of the bacterial fermentation of undigested dietary fiber in the human large intestine. Evidence to date implicates propionate/butyrate as inhibitors of colon cancer cell proliferation and inducers of apoptotic cell death. In this study, we demonstrated that propionate/butyrate was able to induce another type of programmed cell death named autophagy in human colon cancer HCT116 cells. Propionate/butyrate-treated cells exhibited extensive characteristics of autophagy, namely the conversion of LC3-I into LC3-II, reduced expression of p62/ SQSTM1; redistribution of GFPLC3 fusion protein from a diffuse pattern towards autophagosomes, which became visible as cytoplasmic puncta; and increased acidic vesicular organelle development as manifested by dramatically increased quantity of strongly stained punctate areas reflected by LysoTracker staining and monodansylcadaverine (MDC) incorporation. Propionate/butyrate-induced autophagy was based on inhibition of its negative regulator-mTOR pathway as evidenced by dephosphorylation of p70S6K and 4E-BP1 and was marked by increased phosphorylation of AMP kinase, two events known to be linked to autophagy. Activation of AMPK was associated with a fluctuation of intracellular ATP, during which the level of ATP transiently increased following propionate/butyrate exposure, reaching maximum at 5 hours, and then declined in slow but gradual manner. The decline of ATP may be due to compromised mitochondria membrane potential and enhanced cellular reactive oxygen species generation. We also showed that this autophagic increase was associated with a series of changes in metabolic pathways, in which energy-demanding program (fatty acids composition and glycogen synthesis inhibition) was downregulated whereas energy-producing program (mitochondria biogenesis) was initiated to cope with the energy crisis. Finally, we showed that increased autophagy and mTOR inhibition by propionate/butyrate are not dependent on p53 activation. Altogether, this study demonstrates autophagy as a novel programmed cell death pathway for propionate/butyrate induced cell death.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C8.
Abstract C84: Inhibition of cholesterol esterification by fenretinide in osteosarcoma in vitro
Authors: Gasperi-Campani Anna , Roncuzzi Laura , Baiocchi Daniela , Putzolu Marirosa , Baldini Nicola , Giunti Armando , Dessì Sandra
Published: June 2012

Osteosarcoma remains the most common primary malignant bone cancer affecting children and adolescents. Although the combination of systemic chemotherapy and surgery enables long-term survival in most cases, the poor prognosis of patients with metastatic or recurrent disease and the lack of establishment of second-line chemotherapy suggest that novel therapeutic strategies for this tumor are needed. Here we show that fenretinide, a synthetic derivative of all-trans-retinoic acid, is active against osteosarcoma in vitro, at concentrations identical or lower to those detectable in breast cancer patients plasma during chemopreventive clinical trials. Cell lines were HOS and MG63, known to be resistant to methotrexate and cyclophosphamide, drugs in use in osteosarcoma treatment. We demonstrate for the first time that fenretinide is active in this tumor and that the molecular basis of fenretinide activity is the inhibition of cholesterol esterification, with down-regulation of ACAT and MDR1 mRNA, followed by downregulation of caveolin-1 protein expression. This result is in line with our recent results obtained in VSMC and in leukemia cells. The confirmation and extention of these data to different human tumors will provide evidence of a novel mechanism of either cancer or human vascular proliferation disease control by the inhibition of cholesterol esterification. Grants from MIUR, Carisbo Foundation, Pallotti's Legacy for Cancer Research and University of Bologna.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C84.
Abstract C88: Imiquimod induces reactive oxygen species- and caspase-independent cell cycle arrest and apoptosis in MDA-MB-468 breast cancer cells
Authors: Greenshields Anna L. , Hoskin David W.
Published: June 2012

Imiquimod is an immune modulator currently used as a topical treatment for superficial basal cell carcinoma. Recently, apoptosis-inducing activity in neoplastic cells has been attributed to imiquimod at doses over a 1000-fold lower than those used in topical applications. The present study examined the direct effect of imiquimod on MDA-MB-468 breast cancer cells. Oregon Green 488 staining of imiquimod-treated MDA-MB-468 cells indicated that imiquimod had a dose-dependent, anti-proliferative effect; cell cycle analysis indicated that arrest occurred in S phase after 72 h exposure to imiquimod. In addition, imiquimod had a time- and dose-dependent cytotoxic effect on a panel of breast cancer cells (MDA-MB-468, MDA-MB-231, MCF-7, and T47D). Double staining of imiquimod-treated MDA-MB-468 cells using Annexin-V-FLOUS and propidium iodide indicated that imiquimod caused apoptosis, while the lack of lactate dehydrogenase release confirmed the absence of necrosis. Unlike previously reported data for other neoplastic cells, imiquimod-induced apoptosis was not caspase-dependent in MDA-MB-468 cells; however, DiOC6 staining revealed destabilization of the mitochondrial membrane following imiquimod treatment. Pretreatment with reduced glutathione did not rescue MDA-MB-468 cells from imiquimod-induced apoptosis, indicating that reactive oxygen species were not required for imiquimod-induced cytotoxicity. Western blotting showed that imiquimod-treated MDA-MB-468 cells had increased cytosolic cytochrome c and PARP cleavage compared to controls, which was consistent with induction of the mitochondrial pathway of apoptosis. These data suggest that imiquimod exerts a significant caspase-independent apoptosis-promoting effect on breast cancer cells.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C88.
Abstract C98: Blocking glycolysis with 2-deoxy-D-glucose prodrugs to target brain tumors: A novel concept
Authors: Priebe Waldemar , Cybulski Marcin , Fokt Izabela , Skora Stanislaw , Conrad Charles A. , Madden Timothy
Published: June 2012

Glycolysis is the major energy producing pathway for fast growing, glycolytically dependent tumors, such as gliomas and ependymomas. Blocking glycolysis is, therefore, an important therapeutic strategy when used alone or as a combination therapy to enhance the effects of chemotherapy in energy-starved tumors. In our previous studies, we examined D-glucose-based antimetabolites, including 2-deoxy-D-glucose (2-DG), and confirmed their ability to block glycolysis, discovered that they can induce autophagic cell death in vitro, and established their antitumor activity in vivo in an orthotopic glioma model.

However, during our evaluation of 2-DG, we determined that these compounds do not possess sufficient drug-like properties (reasonable biostability, pharmacokinetic characteristics, or distribution to the target) to warrant further investigations into their clinical effectiveness. In an attempt to improve the pharmacokinetic profile of 2-DG, we have designed synthesized, and tested a series of ester-type prodrugs that enhance biostability, biodistribution, and delivery of 2-DG to its CNS target. We will present the synthesis and preliminary evaluation of these compounds, leading to the selection of WP1122 as our lead compound for further drug development. We will also present the subsequent evaluation of WP1122 in vivo in orthotopic brain tumor models and demonstrate its superior pharmacokinetic profile and enhanced CNS uptake.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C98.