bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C44: Positive interaction between Lapatinib and Capecitabine in human breast cancer models: In vitro study of molecular determinants
Authors: Chefrour Mohamed , Fischel Jean-Louis , Giacometti Sarah , Formento Patricia , Renee Nicole , Milano Gerard , Ciccolini Joseph
Published: June 2012

Background: Lapatinib (Tykerb®) is an innovative small molecule with a dual-TKI inhibitory action against both HER1 and HER2. Lapatinib has demonstrated antiproliferative efficacy in various models, including breast tumors, thus heightening the expectations for new breast cancer therapies. The association of Lapatinib with Capecitabine (Xeloda®) has been recently approved, both in the USA and in Europe, in patients with HER2+ metastatic breast cancer, relapsing after trastuzumab treatment. However, little is known on the exact molecular mechanisms underlying the increase in efficacy expected when combining these drugs.

Methods: In the following preclinical study, we studied the molecular determinants of this new targeted drug / antimetabolite association on a panel of representative human breast cancer cell lines: BT474, MCF-7, MDA-MB-468, SK-BR-3, T47D and ZR-75. Effects of concomitant and sequential drug combinations on proliferation and apoptosis were measured, along with the study of the determinants of response/resistance to Capecitabine (e.g., dihydropyrimidine dehydrogenase (DPD), thymidylate synthase (TS), and thymidine phosphorylase (TP) levels), to Lapatinib, (e.g., members of the HER1/2 signaling pathway), and to proliferation (P21) and apoptosis (Bax, Bcl-2).

Results: Additive to moderate synergistic effects were demonstrated (Chou & Talalay combination indices, CI) with CI's ranging between 0.55 and 1.03. Further experiments were performed to identify the mechanisms underlying the efficacy of the combination. We observed that Lapatinib down-regulated thymidylate synthase (−30%), thus suggesting a better cytotoxic effect of Capecitabine when tumor cells were preliminary exposed to Lapatinib, but had not effect on DPD activity. Impact on TP levels was model-dependent, but could lead additionally to a better activation towards active metabolites eventually. Besides, adding Capecitabine to Lapatinib strongly down-regulated P-AKT (e.g., from −56 to −100%) and p-P42/44 (−78 to −100%), allowing a deeper inhibition of the HER1 and HER2 downstream signaling pathway. A +150 to +300% increase of the Bax/Bcl2 ratio in cells exposed to both drugs was observed, leading to the enhancement of apoptosis and cell death. Those findings were consistent with the observation that the association of Lapatinib and Capecitabine led to an increase of P21 protein expression and consequently to cell cycle arrest.

Conclusion: Taken together, these results suggest that the more-than-additive effects observed when associating Lapatinib and Capecitabine is mostly based both on TS modulation and HER1 and HER2 signaling pathways down-regulation, eventually resulting in apoptosis and cell death. In vivo experiments are ongoing to fully confirm these data.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C44.
Abstract C48: The role of the neuregulins and their receptors in the islets of Langerhans
Authors: South Jack C. M. , Gullick William J.
Published: June 2012

The Neuregulins are a sub-class of the Epidermal Growth Factor (EGF) family of mitogenic ligands. This research aims to investigate their expression in individual cell types within the Islets of Langerhans in order to determine whether they play a role in regulating islet cell growth or hormone secretion. This information may be useful in the future in treating diabetic patients or in rare instances of islet cell cancer.

An expression profile of all the EGF receptors and ligands, including the four NRG genes and some of their splice variants, was determined in rat islets using immunohistochemical staining with specific mouse monoclonal and rabbit polyclonal antibodies. Double immunofluorescent staining with antibodies to the ligands or receptors and to the main islet cell hormones, insulin, glucagon and somatastatin was used to identify which cell types were expressing each isoform. The rat islet tumor cell line CRI-G1 was selected as a model system to determine cellular responses to addition of the different ligands. Immunofluorescent staining and RT-PCR was used to construct a receptor and ligand expression profile.

Immunohistochemistry demonstrated that some ligands such as NRG3 or the alpha and beta isoforms of NRG1 were expressed in restricted regions of the islets. Through double immunofluorescence it was found that NRG-1α was present in β-cells and NRG-1β and NRG-3 were mainly present in α-cells. The same restricted pattern of expression was also found in human islets. In contrast, staining for HER-3 and HER-4 receptors in rat islets was much more homogenous. In using single immunofluorescence with the CRI-G1 cell line both HER-3 and HER-4 as well as with two of the NRG-4 splice variants were detected. PCR using cDNA from the cell line confirmed the expression of both NRG-1 and NRG-2 at the mRNA level.

Differential expression of NRG-1α and 1 in or cells due to alternative splicing may play a role in regulating cell proliferation, differentiation or hormone secretion in individual islet cell types across a range of species. Differences in localisation between the three Neuregulin forms and their two receptors suggests that both autocrine and paracrine neuregulin-mediated signalling occurs. The expression of NRG-1, NRG-2, NRG-4 and the receptors HER-3 and HER-4 within the CRI-G1 cell line provides a model system to explore the activity of this complex system in vitro.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C48.
Abstract C58: Causal Network™ Modeling identifies common and unique mechanisms for sensitivity to the PI3K inhibitor GSK1059615 and the AKT inhibitor GSK690693
Authors: Macoritto Michael P. , Laifenfeld Daphna , Catlett Natalie , Jorge Milena , Matthews Andrea , Auger Kurt R. , Kumar Rakesh
Published: June 2012

Background: Aberrant PI3K and AKT signaling play an important role in the development of some human tumors by promoting cell proliferation and survival. Inhibitors that target the kinase activity of PI3K or AKT may constitute an effective treatment for human malignancies that rely on these molecules for their growth. GlaxoSmithKline has developed a PI3K inhibitor, GSK1059615, and a pan-AKT inhibitor, GSK690693. Since AKT is activated by PI3K via generation of PIP3, and is a key effector of PI3K signaling, a high degree of overlap is expected in the effects of a PI3K and an AKT inhibitor. To determine whether the effects of the PI3K inhibitor are mediated solely through the AKT pathway, we compared the molecular response of BT474 human breast cancer cells to both GSK1059615 and GSK690693 as well as the sensitivity profile of 16 other human cancer cell lines.

Methods: The mechanisms of action of the PI3K inhibitor GSK1059615 and the AKT inhibitor GSK690693 were evaluated in BT474 human breast cancer cells using RNA expression and phosphoproteomic data. Causal Network™ Modeling was used to characterize these data sets to identify processes that contribute to the inhibition of proliferation observed in cells sensitive to these inhibitors, and processes that could explain differences in sensitivity to the two inhibitors. The sensitivity/resistance profile of 16 human cancer cell lines to GSK1059615 and GSK690693 was also determined.

Results: Four common processes were found in BT474 cells treated with either GSK690693 or GSK1059615: RB1-Mediated Cell Cycle Arrest

Increased FOXO Signaling

Decreased MYC and TFRC Signaling

Decreased Metabolism

We identified one process specific to the PI3K inhibitor GSK1059615-treated cells: Decreased MAPK Signaling

Conclusions: In BT474 cells, GSK690693 and GSK1059615 inhibit cell proliferation by increasing RB1-mediated cell cycle arrest, increasing FOXO signaling, decreasing MYC signaling, and decreasing cellular metabolism, as identified by Causal Network™ Modeling. In addition to these four shared mechanisms, the PI3K inhibitor GSK1059615 attenuates MAPK signaling. Five of 16 cell lines tested for sensitivity to the inhibitors were sensitive to GSK1059615 treatment only, and these five cell lines were the only ones that contained mutations activating MAPK signaling. Thus decreased MAPK signaling is likely an additional mechanism by which GSK1059615 inhibits cell proliferation, and may confer sensitivity to this PI3K inhibitor in cells resistant to the AKT inhibitor.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C58.
Abstract C77: In vivo anticancer activity of an aurora kinase inhibitor, VX-680, in gastric cancer
Authors: Kim Hyo Song , Yu Eun Jeong , Jeung Hei-Cheul , Kim Tae Soo , Chung Hyun Cheol , Roh Jae Kyung , Noh Sung Hoon , Jang Paul , Kim Minkyu , Rha Sun Young
Published: June 2012

Background: Aurora kinases (AK) constitute one family of seine/threonine kinases, whose activity is essential for mitotic progression. Expression of aurora kinase is greatest in G2-M phase, and their function includes spindle formation, centrosome maturation, chromosomal segregation, and cytokinesis. Over-expression of AK is related to aneuploidy and carcinogenesis. Herein, we investigated the in vitro and in vivo anticancer acvitivity of a novel AK inhibitor, VX-680 in gastric cancer.

Methods: AK protein expression and kinase activity were screened in gastric cancer cell lines using immunoblot and in vitro kinase assay, respectively. The anti-proliferative activity of VX-680 was measured using MTT assay. The changes of cell cycle by the treatment of VX-680 were analyzed by flow cytometry. YCC-16 cell line selected for tumor xenograft models. In vivo activity validation using mouse xenograft models and extract protein or tissue slide from each tumors.

Results: AK protein expression and its kinase activity were variable among cell lines. VX-680 showed anti-proliferative effect in vitro on a wide range of gastric cancer cell lines, and calculating the IC50 value, it was distributed from 0.04uM to 19.87uM. It was correlated with kinase activity, not with protein expression of AK. VX-680 induced the reduction of phosphor-histone H3 and the cell cycle defects that accumulated >4N DNA. In vivo experiment, confirmed that AKI had an effect on tumor inhibition. Solid tumor formed with YCC-16 cell lines and VX-680 was injected twice a day for 5 days with a dose of 50mg/kg. Estimating the tumor volume for 21 days after VX-680 injection, the difference in tumor volume between the control and the treated mice were statistically significant. Kinase activity and phospho-histone H3 in VX-680 treated tumor was reduced in drug treatment samples compared to the control samples.

Conclusion: We demonstrated that VX-680 has anti-cancer activity in gastric cancer. Kinase activity of AK might be a predictor for sensitivity of VX-680.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C77.
Abstract C78: The mu opioid receptor regulates Lewis lung carcinoma tumor growth and metastasis
Authors: Mathew Biji , Lennon Frances E. , Siegler Jessica , Gerhold Lynnette , Mambetsariev Nurbek , Moreno-Vinasco Liliana , Garcia Joe G.N. , Salgia Ravi , Moss Jonathan , Singleton Patrick
Published: June 2012

Introduction: Expression of the mu opioid receptor (MOR) is increased in patients with non-small cell lung cancer (NSCLC), a disease with poor prognosis and limited therapies. Previous work by our laboratory and others suggested a role of opioids in several aspects of tumor growth (Anticancer Res 2009;29:3195–3205, Mol Cancer Ther 2008;7:1669–1679, Cancer Res 2002;62:4491–4498). In this study, we investigated MOR as a potential therapeutic target for lung carcinoma.

Methods: Lewis lung carcinoma (LLC) cells were treated with MOR siRNA or the peripheral MOR antagonist, methylnaltrexone (MNTX, 250 nM), prior to the addition of EGF (10 ng/ml), IGF (10 ng/ml), DAMGO (1 nM), morphine (1 nM) or serum (1, 5 or 10%). In vitro functional (cell proliferation and invasion) and biochemical studies (immunoblotting) were then conducted. For in vivo assays, C57BL/6J mice were injected intravenously with dual color (GFP-RFP labeled) LLC cells, with or without MOR siRNA treatment. In another experiment, MOR knockout as well as wildtype control mice were injected with LLC cells (subcutaneously into the flank) and followed for 12 weeks. In a parallel experiment, LLC tumor bearing C57BL/6J mice received continuous infusion of MNTX (10 mg/kg/day) for 2 weeks. Tumor growth and lung metastasis were evaluated using tumor volume measurements and/or in vivo fluorescent microscopy (Olympus OV-100) with or without intravenous injection of ProSense and/or MMP Sense probes.

Results: Our data indicate that LLC cells have ∼5 fold increased expression of MOR (compared to primary lung epithelial or BEAS-2B cells). Inhibition of MOR with siRNA or MNTX treatment reduced in vitro LLC proliferation (90%) and invasion (50–75%). In vivo lung metastasis was reduced by ∼75% in wildtype C57BL/6J mice with LLC lacking MOR (siRNA) versus control siRNA-treated LLC (3 weeks post-i.v. injection, quantitated with OV-100). In addition, primary LLC tumor volume was substantially reduced (>90%) in MOR knockout mice compared to wildtype mice (flank injection). Finally, we observed that continuous infusion of MNTX (10 mg/kg/day) in mice with pre-existing LLC primary tumors attenuates further tumor growth.

Conclusion: We have shown that MOR is a potential therapeutic target for inhibition of LLC proliferation, invasion and metastasis. In addition, MNTX can attenuate LLC primary tumor growth. Further study of MNTX as a potential therapeutic agent is warranted.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C78.
Abstract C83: The proliferation marker Ki-67 as novel molecular target in cancer therapy
Authors: Rahmanzadeh Ramtin , Rai Prakash , Rizvi Imran , Celli Jonathan , Abu-Yousif Adnan , Baron-Luehr Bettina , Gerdes Johannes , Hasan Tayyaba
Published: June 2012

Introduction: For patients with advanced ovarian cancer there are no effective therapies. The need for alternative treatments with high specificity but with low systemic toxicity can be achieved by targeting key molecular markers associated with cancer cells. Here, we show an innovative proof-of-principle approach for efficient killing of proliferating ovarian cancer cells by inactivating a protein associated with cell proliferation namely, the nuclear Ki-67 protein (pKi-67), using nanotechnology-based photodynamic therapy (PDT). pKi-67 is highly expressed in all proliferating cells and antibodies against this protein are widely used as prognostic tools in tumor diagnosis.

Materials and Methods: Anti pKi-67 antibodies were first conjugated to fluorescein isothiocyanate (FITC) and then encapsulated inside liposomes. The liposomes encapsulating antibodies were characterized by dynamic light scattering and transmission electron microscopy. We then evaluated the efficacy of Ki-67 targeting using two in vitro ovarian cancer models - monolayer cultures as well as 3D cultures established in our lab. Cell viability after irradiation with laserlight at 488 nm was assessed using MTT viability assay for monolayer cultures while a Live/Dead assay kit was used for analyzing 3D cultures.

Results: After incubation of OVCAR-5 ovarian cancer cells with the liposomal constructs, we used confocal microscopy to image localization of the antibodies to the nucleoli of the cells. Irradiation of these cells with a 488 nm laser led to a significant loss of viability. The efficacy of this approach was further demonstrated in a 3D culture model of OVCAR-5 cells. Incubation of 3D cultures with the liposomal constructs followed by light irradiation led to destruction of their acinar structures over 72 h following treatment. Using two different anti pKi-67 antibodies, where one targets the “active” form of pKi-67 while the other binds to the “inactive” form of the protein; we show that cell killing is specific after inactivation of the “active” Ki-67 protein. Further, the specificity of this approach for pKi-67 positive cells is demonstrated in confluent human lung fibroblasts (MRC-5) where minimal cell death was observed. This is in agreement with the flow cytometry results which show that only small populations of confluent MRC-5 cells express pKi-67.

Conclusions: Taken together, our findings suggest that pKi-67 is an attractive therapeutic target in cancer and this approach holds promise as an effective alternative cancer therapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C83.
Abstract A108: LY2606368, a second generation Chk1 inhibitor, inhibits growth of ovarian carcinoma xenografts either as monotherapy or in combination with standard-of-care agents.
Authors: McNeely Samuel C. , Burke Teresa F. , DurlandBusbice Sara , Barnard Darlene S. , Marshall Mark S. , Bence Aimee K. , Beckmann Richard P.
Published: June 2012

Chk1 is a key component of the cellular response to DNA damage resulting from exposure to various genotoxic agents. When damaged DNA is present, Chk1 maintains genomic integrity by stalling cell cycle progression thereby providing time for the appropriate repair to occur. Inhibition of Chk1 kinase activity leads to a loss in the regulation of this damage response and results in abrogation of cell cycle arrest and ultimately to cell death as cells with damaged DNA proceed inappropriately into mitosis. Thus first generation small molecule inhibitors of Chk1 kinase were developed to act as companion compounds that would be used together with established standard -of-care (SoC) cancer treatments that target DNA to enhance the effectiveness of such treatments. As a second generation inhibitor, LY2606368 distinguishes itself from other molecules in this class in that it has been shown to inhibit tumor growth when used either as monotherapy or in combination with SoC agents. The single agent activity of LY2606368 is likely related to the ability of this molecule to potently inhibit Chk1 thereby effectively interfering with its function to support normal DNA replication via regulation of origin firing and/or aspects of chromosomal dynamics during mitosis which are thought to be dependent upon Chk1 kinase activity. The studies presented here have evaluated the potential of LY2606368 to inhibit ovarian tumor growth both as monotherapy and in combination with cisplatin or paclitaxel. Treatment of tumor-bearing animals on a BID X 3 schedule with LY2606368 alone resulted in profound growth inhibition such that greater than 80% inhibition was observed at doses of 12mg/kg in both subcutaneous and orthotopic models. Moreover, the studies in the orthotopic SKOV3 model demonstrated that treatment with LY2606368 not only robustly inhibited growth of the primary tumor but also significantly reduced the incidence of metastases and accumulation of ascites fluid. Subsequent studies in SKOV3 subcutaneous xenografts revealed that significant growth inhibition could be achieved with various combination strategies which include agents that are commonly used in the treatment of ovarian cancer. In particular, the extent of growth inhibition that could be achieved with cisplatin or paclitaxel alone was augmented by combining these agents with LY2606368. Overall these animal studies indicate the potential therapeutic benefit in ovarian cancer that could be achieved with LY2606368 either as monotherapy or in combination with SoC agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A108.
Abstract A123: TEL is a dual anticancer agent that targets Stat3 and HIF-1α.
Authors: Chen Han , Guan Yongli , Gu Yuan , Tweardy David , Jing Naijie
Published: June 2012

We recently developed a dual anti-cancer agent, TEL (a perylence derivative) that independently targets both Stat3 and HIF-1α. Stat3 and HIF-1 have been demonstrated to be important targets for cancer therapy. Stat3 participates in oncogenesis through the up-regulation of genes encoding anti-apoptosis, cell-cycle regulators, and inducers of angiogenesis. Also, Stat3 suppresses anti-tumor immune responses and promotes inflammation-induced cancer, making it an attractive target. HIF-1 mediates the cellular response to hypoxia and activates the transcription of genes that are involved in angiogenesis, cell survival, glucose metabolism and invasion. HIF-1α was demonstrated to be overexpressed in many human cancers. Overexpression of HIF-1α, which results from intratumoral hypoxia and genetic alternations, has been associated with poor prognosis and treatment failure in a number of cancers. Our previous results demonstrated that targeting both Stat3 and HIF-1α together could improve tumor response, compared to either single agent alone, and reduce drug resistance and treatment failure.

Recently, we have isolated or derived more than 20 molecules from Chinese medicinal herbs. Based on drug screening, we found that TEL selectively targets both HIF-1α and Stat3, significantly inhibiting activation of HIF-1α and p-Stat3, and blocking the expression of both HIF-1α and Stat3 regulated oncogenes (e.g. Bcl2, VEGF, Glut1, and others) in cancer cells, showing that it has great potential to be an anti-cancer agent. Our results demonstrated that (1) TEL inhibits both p-Stat3 and HIF-1α; (2) TEL strongly interacts with the residues of E612, S613 and E638 in the SH2 domain of Stat3 and disrupts the interaction between Stat3 and the phosphotyrosine-stimulating receptor, blocking Stat3 phosphorylation; (3) TEL directly binds to HIF-1 protein and inhibits the interaction between N803 of HIF-1α and p300/CBP under hypoxia condition, inducing the proteasomal degradation and blocking its transcription; and (4) TEL greatly induces cancer cell apoptosis by targeting Stat3 and HIF-1α, but does not cause toxicity in normal cells under the same condition. TEL is likely to have a favorable safety profile, as do many Chinese herbed medicines. Therefore, TEL not only has broad drug activity for cancer therapy, but may be well tolerated in cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A123.
Abstract A125: Silencing Stat3 signaling in human cancers: Identifying potent small molecule inhibitors of Stat3 function.
Authors: Page Brent D. G. , Zhang Xialoei , Atkinson Jennifer , Li Zhi Hua , Schimmer Aaron D. , Trudel Suzanne , Turkson James , Gunning Patrick T.
Published: June 2012

Stat3 is essential for transducing signals from extracellular stimuli, but also functions as a nuclear transcription factor required for regulating genes involved in proliferation, apoptosis, angiogenesis and invasion, in addition to genes encoding cytokines, chemokines and growth factors. In contrast to the transient nature of Stat3 activation in normal cells, many human cancers, including breast, prostate, ovarian, brain and multiple myeloma (MM) harbor constitutive Stat3 activity. Stat3 downstream target genes are critical to the dysregulated biological processes that promote tumor cell growth, survival and induce chemoresistance, thus targeting Stat3 signaling represents an important therapeutic target in cancer therapy.

We have rationally designed and developed Stat3 inhibitors that disrupt transcriptionaly active Stat3-Stat3 homo-dimers, suppress Stat3 activation (phosphorylation), inhibit Stat3-target gene expression (c-Myc, Bcl-xL, survivin) and potently induce apoptosis in tumor cells harboring aberrant Stat3 activity. Moreover, lead compound BP-1–102, a salicylic acid containing small molecule, induced strong antitumor effects on human breast cancer (MDA-MB-231) xenografts and in MM preclinical tumor models. Most notably, given via oral gavage, BP-1–102 strongly inhibited the growth of human breast tumor xenografts, identifying it as a most potent orally bioavailable Stat3-targeting inhibitor.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A125.
Abstract A130: Fragment-based discovery and optimization of Rho kinase inhibitors.
Authors: Liu Yan , Wang Binglin , Patel Ronil A. , Sun Nan , Sung Shen-Shu , Pireddu Roberta , Lawrence Nicholas J. , Sebti Said M. , Li Rongshi
Published: June 2012

Using high concentration biochemical assays and fragment-based screening, we designed and optimized a novel class of Rho-kinase inhibitors. Ligand Efficiency (LE) was employed to assess the binding potential of the fragments and to guide the optimization process. In addition, molecular modeling was used to aide the design of potent inhibitors. A series of fragments as hinge binders were designed and synthesized, and screened using high concentration biochemical assays. Both the spacer length and isomeric tail molecules played key roles in both activity and selectivity. Structure activity relationship studies led to optimization of fragments that yielded potent and selective ROCK 1 and ROCK 2 inhibitors. For instance, compound 24 was highly potent and selective for ROCK 2 (IC50 = 100 nM) over ROCK 1 (IC50 = 1690 nM) whereas compound 18 was potent and selective for ROCK 1 (IC50 = 460 nM) over ROCK 2 (IC50 = 700nM). Compound 24 but not its inactive stereoisomer 23, and compound 18 but not its inactive analog 11 inhibited the phosphorylation of Rho Kinase substrates in intact human cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A130.
Abstract A131: Drugging the telomere: A rational approach for the selection quadruplex DNA targeting agents for the treatment of cancer.
Authors: Hummersone Marc G.
Published: June 2012

The pentacyclic acridinium class of small molecule telomere targeting agents have been shown to stabilise the 3'single-stranded guanine-rich telomeric overhang to fold into a G-quadruplex structure. Stabilisation of the latter is incompatible with an attachment of telomerase and a collection of protective proteins referred to as the shelterin complex to the telomere. G-quadruplex ligands can effectively inhibit the telomere maintenance functions of telomerase while revealing the chromosomal termini to the unwanted attention of DNA repair proteins resulting in telomere specific damage. Potent antitumor properties of the G-quadruplex ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) have been reported and shown to be driven by telomere uncapping events. However, in preliminary toxological studies the original lead compound (RHPS4) revealed cardiovascular liabilities related to interactions with hERG receptor (IC50 0.2 uM) as well as additional receptor interactions including muscarinic M1, M2 and M3 and Beta2 adrenergic receptors.

Pharminox have designed and synthesised second generation agents with significantly improved on and off target profiles. Here we describe the modification and optimisation of new telomere targeting agents with an emphasis on their specific effect on telomeres.

Specific bio-molecular assays have been performed on the best compounds of the series, as defined by telomeric DNA binding characteristics and cancer cell activities, “normal” cell selectivities and off target affinities. It was found that all new compounds examined were more potent than RHPS4 as telomere damage inducers. Specifically, at the dose of 0,1 uM, the compounds PMX-000177-761-785 trigger a percentage of TIF-positive cells comparable to that induced by RHPS4 at a 5 fold higher drug concentration. Activation of damage response mainly occurred in replicating cells for the compounds PMX-000177 and -785, suggesting that they can work with a RHPS4-like mechanism of action. As part of a defined screening cascade and candidate target profile, these studies are being used to differentiate between compounds as they progress towards pre-clinical candidate selection status.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A131.
Abstract A132: S1PR5 structural model and its application in drug discovery.
Authors: Sung Shen-Shu
Published: June 2012

Sphingosine-1-phosphate (S1P) is a bioactive lipid with diverse functions, including cell proliferation, differentiation, angiogenesis, chemotaxis, and migration. Many of these activities are mediated through five closely related G-protein-coupled receptors (GPCR) of the S1P receptor (S1PR) family. Because of the technical challenges in crystallization of membrane-bound proteins, S1PR structures have not become publicly available. Instead, homology models were built based on a theoretical model of the retina pigment rhodopsin, which is a GPCR with a covalently bound ligand (Parrill, A. L. et al. J. Biol. Chem. 2000, 275, 39379). Recently, new techniques in crystallization have made more GPCR structures available, including those in complex with their agonist, antagonist, or inverse agonist. These structures provide more realistic basis for building S1PR structural models, and at the same time raise a new question about which structure to use in drug design and virtual screening. In this presentation, two structural models of S1PR subtype 5 (S1PR5, Kothapalli, R, Kusmartseva I, and Loughran Jr T. P. et al. Biochim. Biophys. Acta 2002, 1579, 117) are constructed based on crystal structures of β2-adrenergic receptor (β2AR), one of the most extensively studied GPCR. A nanobody-stabilized active state of β2AR (Soren, G. F. et al. Nature 2011, 469, 175) and an inactive state of β2AR in complex with its partial inverse agonist (Cherezov, V. et al. Science 2007, 318, 1258; Hanson, M. A. et al. Structure 2008, 16, 897) are used in building the two models, which are subsequently tested by in silico docking of their known ligands. The models based on active and inactive β2AR are used in virtual screening to find potential activators and inhibitors, respectively. Results from the two models are compared and their different applications are discussed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A132.
Abstract A139: Development of EML4-ALK addicted cell models for evaluating sensitivity to ALK inhibitors.
Authors: Ise Nobuyuki , Nambara Daisuke , Omi Kazuya , Goishi Katsutoshi
Published: June 2012

Background: EML4-ALK fusion tyrosine kinase has been implicated in the pathogenesis of a subset of non-small-cell lung cancers (NSCLCs). The fusion kinase is a rational therapeutic target for addicted cancer cells whose sustained proliferation and survival depend on it. Cancer cell lines harboring the fusion kinase gene are useful for screening effective therapeutic agents. However, few cell lines have been identified so far, and these cell lines do not represent all the variants discovered in clinical specimens. Moreover, drug-resistant mutations in the fusion gene have recently been identified in patients treated with ALK inhibitors. Techniques to establish cell models addicted to certain genes will be invaluable for developing effective drugs for molecular targeted therapy. We have developed a new method to easily and efficiently establish cell models exhibiting addiction to introduced oncogenes. We evaluated the method for variants of EML4-ALK fusion oncogene including drug-resistant mutants.

Material and methods: EGFR-addicted NSCLC cell lines were transfected with each variant of EML4-ALK fusion gene, and the cells addicted to the introduced fusion gene for their growth and survival were selected. Expression of EML4-ALKs in the obtained cells was confirmed by immunoblotting. Their dependence on the introduced fusion gene was assessed by MTS assay following ALK inhibitors treatment. Direct inhibitory effect of the drugs was analyzed by monitoring the autophosphorylation levels of EML4-ALKs.

Results: EML4-ALK addicted cells were efficiently established from all the transfectants. They expressed the introduced variant of EML4-ALK. Each of the ALK inhibitors tested in this study exerted distinct anti-growth activities toward the established cell models. PF-2341066 had the strongest, dose-dependent inhibitory effect on the growth of the addicted cells. This agent also inhibited autophosphorylation of EML4-ALKs in parallel with the growth inhibition activity, but failed to inhibit cells expressing drug-resistant EML-4-ALK.

Conclusions: We demonstrated that cancer cell models addicted to EML4-ALK can be easily established from EGFR-addicted cell lines. Since the established cells depended on the introduced EML4-ALK genes for their growth and survival, the introduced genes likely preserved their oncogenic functions in these cells. Our method will provide useful cell models to evaluate the efficacy of various inhibitors in vitro and elucidate functions and signaling pathway of EML4-ALKs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A139.
Abstract A140: Development of high-throughput assay for new agents that inhibit the transcription of the HIF-1alpha gene.
Authors: Sun Daekyu , Shin Yoon-Joo , Uribe Diana
Published: June 2012

The hypoxic (low-oxygen) region is developed in most solid tumors as they grow and outstrip their blood supply. A critical mediator of this series of hypoxic responses is known as the transcription factor hypoxia-inducible factor 1 (HIF-1), which mediates increased expression of proteins that promote angiogenesis, anaerobic metabolism, and many other survival pathways. Therefore, HIF-1 alpha has been proposed as a particular target for therapeutic intervention of human cancer. Thus, there is need for extensive discovery of new types of more specific HIF-1 inhibitors, including small molecules that directly inhibit the transcription of the HIF-1 alpha gene. In our preliminary studies, we have demonstrated that the transcription of HIF-1 alpha gene is greatly dependent upon the presence of the pPu/pPy tract within its proximal promoter region. We further explored if it is feasible to identify small molecules that inhibit the transcription of the HIF-1 alpha gene by interfering with the assembly of the transcription machinery to the proximal promoter region of this gene. As a means of measuring promoter response to small molecules in cells, the luciferase reporter-based cell assays would be simple, straightforward, and very effective. Thus, this assay could also be used to identify transcriptional inhibitors of the HIF-1 alpha gene. To determine if luciferase reporter assay is amenable for high-throughput screening for novel inhibitors of the transcription of the HIF-1 alpha gene, we used the Flp-In system to generate genetically engineered Flp-In 293 cell lines stably expressing the firefly luciferase gene under the control of the HIF-1 alpha promoter as described in our preliminary studies. In our preliminary studies, we have also demonstrated that the transcription of HIF-1 alpha gene is greatly dependent upon the presence of the pPu/pPy tract within its proximal promoter region.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A140.
Abstract A141: Analysis of cellular kinase activity of resistance-related MET mutants using transmembrane-domain (TMD) activated kinase variants.
Authors: Weber Holger , Mueller Daniel , Totzke Frank , Feger Daniel , Mueller Melanie , Birkle Marianne , Kubbutat Michael HG , Schaechtele Christoph , Ballmer-Hofer Kurt , Ehlert Jan E. , Graeser Ralph
Published: June 2012

Kinases are important targets in oncology. However, the frequent emergence of resistance (gatekeeper) mutants in target kinases seriously limits the patient's response time to a given drug. Secondary drugs need to be developed that specifically target mutant enzymes. The discovery of such kinase inhibitors requires relevant assay systems. In order to efficiently analyze whether novel inhibitors target gatekeeper mutants of a given kinase in a cellular context we set out to establish a suitable cellular test system for the receptor tyrosine kinase MET. Our aim was to minimize variations such as cellular background and ligand dependent activation while putting the focus on the mutation of interest. In an approach to uncouple kinase activity from ligand activation, we screened a set of artificial transmembrane domains (TMD) to identify a TMD that induces constitutive MET autophosphorylation in transient transfections. Subsequently, Rat1 cells were transduced to express the tag-labelled intracellular wild-type (wt) MET domain harbouring the activating TMD while lacking the extracellular domain. Characterization of TMD-MET-wt in Rat1 cells showed constitutive MET expression and phosphorylation. ELISA studies to analyze the inhibition of MET phosphorylation by cognate MET inhibitors revealed comparable IC50 values to those obtained with wild-type MET naturally over expressed in MKN45 gastric carcinoma cells. Moreover, expression of TMD-MET-wt in Rat1 cells led to cell transformation as assessed by growth in soft agar as well as in vivo as a subcutaneous xenograft. Interestingly, the same artificial TMD induced constitutive activity of various gatekeeper mutants of MET when transduced into Rat1 cells. This observation allowed for the comparative analysis of various small molecule MET inhibitors on multiple MET mutants in an identical cellular background and without the need of ligand-induced kinase activation. Using a straight forward ELISA-based approach, we generated profiles of various MET inhibitors on cellular MET mutant phosphorylation and compared these with inhibition profiles obtained in biochemical MET activity assays.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A141.
Abstract A145: EGFR-targeting of carbon nanovectors loaded with paclitaxel improves their antitumor efficacy and radiosensitization of head and neck squamous cell carcinoma in vivo.
Authors: Sano Daisuke , Berlin Jacob M. , Marcano Daniela C. , Valdecanas David R. , Zhou Ge , Milas Luka , Tour James M. , Myers Jeffrey N.
Published: June 2012

Nanoparticels have been researched broadly as new generation of diagnostics, imaging agents, and drugs for detecting and treating cancer. We previously developed PEGylated small hydrophilic carbon clusters (PEG-HCCs) and reported that PEG-HCCs conjugated with paclitaxel (PTX/PEG-HCCs) is a stable effective drug delivery system with minimal toxicity. Here, we describe the establishment of targeted nanovectors by simply mixing PTX/PEG-HCCs with cetuximab (ImClone Systems), an anti-epidermal growth factor receptor (EGFR) monoclonal antibody. The specificity of this EGFR targeted method of delivery of PTX/PEG-HCCs was demonstrated in mice implanted subcutaneously on opposing flanks with EGFR expressing, OSC-19 (head and neck squamous cell carcinoma: HNSCC) cells and non-EGFR-expressing MCF-7 cells in a nude mouse. The targeted nanovector system, Cet/PTX/PEG-HCCs, showed greater efficacy superior than PTX/PEG-HCCs on OSC-19 tumor growth, but not on MCF-7 tumors. Cet/PTX/PEG-HCCs also showed greater growth inhibition prolonged survival compared to PTX or PTX/PEG-HCCs in an orthotopic nude mouse model of human HNSCC. These results suggested that Cet/PTX/PEG-HCCs can targeted to HNSCC overexpressing EGFR cells. In addition, Cet/PTX/PEG-HCCs were found to radiosensitize HNSCC cells both in vitro and in vivo. Cet/PTX/PEG-HCCs plus radiation inhibited tumor growth and prolonged survival in vivo. This work is highly significant as success would allow for targeted chemotherapy for HNSCC by simply mixing commercially available drugs and antibodies with a nanovector solution.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A145.
Abstract A16: Defining the essential in vivo oncogenic function of p63 in squamous cell carcinoma.
Authors: Ramsey Matthew R. , Wilson Catherine , Ory Benjamin , Faquin William C. , Milla Alea A. , Ellisen Leif W.
Published: June 2012

The p63 transcription factor controls diverse functional programs within the epithelium. Despite its essential developmental role, however, the precise contribution of p63 in cancer remains controversial, and the relevant p63-dependent pathways remain poorly understood. Squamous Cell Carcinoma (SCC) is a common and treatment-refractory form of human cancer that can arise in various epithelial tissues including the lungs, skin, and head and neck region. In SCC, the p63 locus is frequently targeted for genomic amplification and/or overexpression, leading to tumor-specific up-regulation of ΔNp63α. In vitro studies point to a role for ΔNp63α in survival of SCC cells, yet such models are unlikely to fully reflect the contribution of p63 in squamous cancers. We therefore developed an in vivo model in order to address in a definitive manner the role of p63 in progression and maintenance of this disease. We generated a murine model of SCC that faithfully recapitulates the molecular and pathological features of human SCC, including high-level expression of p63, invasion and metastasis. We crossed this model to a conditional p63-null allele (p63flox) and a tamoxifen-inducible Cre transgene, K14-CreER. Remarkably, we find that genetic excision of endogenous p63 in SCC-bearing mice results in rapid, dramatic, and sustained regression of tumors. This effect is due in large part to increased tumor-specific apoptosis, and is associated with no appreciable effects on normal p63-expressing tissues. We then performed global gene expression profiling and extensive validation in order to uncover the key transcriptional programs mediating this survival effect. Unlike previous in vitro work which defined a predominant role for pro-apoptotic Bcl-2 family members in p63-dependent tumor cell survival, our in vivo studies have uncovered a key role for growth factor receptor tyrosine kinases (RTKs) downstream of p63. We find that p63 is a direct transcriptional regulator of these RTK genes, and we support the relevance of these findings to human disease by showing a strong correlation between p63 and these essential p63-regulated genes in human head and neck SCC tumors. Collectively, these studies have established the oncogenic function of p63 in SCC, and they have uncovered a novel and essential pathway mediating p63-dependent tumor maintenance. Ongoing work to be presented seeks to validate this RTK pathway as a potentially important therapeutic target in SCCs which overexpress p63.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A16.
Abstract A178: Developing chemotherapeutics which selectively disable the actin cytoskeleton of tumor cells.
Authors: Stehn Justine R. , Haass Nikolas K. , Bonello Teresa , Desouza Melissa , Kottyan Gregg , Treutlein Herbert , Zeng Jun , Nascimento Paula R.B.B. , Butler Tanya , Hill Timothy A. , McCluskey Adam , Palmer Stephen , Hardeman Edna C. , Winlaw David , Weninger Wolfgang , Cripe Timothy P. , Gunning Peter
Published: June 2012

Background: The actin cytoskeleton is an important regulator of a variety of cellular functions including cell motility, adhesion, and proliferation, making it an ideal chemotherapeutic target. Despite this promise there are still no anti-actin compounds used in current chemotherapy, primarily due to the inability of existing anti-actin agents to discriminate between the actin cytoskeleton of tumor cells and the actin filaments of the muscle sarcomere. We have previously shown that tropomyosin, an integral component of the actin cytoskeleton, defines functionally distinct populations of actin filaments. We have identified a specific tropomyosin isoform common to all tumor cells tested to date which regulates cell proliferation and have designed a new class of compounds to target this filament population.

Summary of Results: Anti-tropomyosin (Tm) compounds were selected based their ability to target the actin cytoskeleton and their efficacy against a panel of neuroblastoma and melanoma cell lines. The lead compound, TR100, was shown to be effective against a panel of tumor cell lines with an average EC50 of 3μM. When tested in 3D melanoma spheroid models, which more accurately mimic the tumor microenvironment, TR100 inhibited melanoma cell growth and motility. This effect translated to a reduction in tumor cell growth in vivo in both neuroblastoma and melanoma xenograft models. In vitro data using isolated rat cardiomyocytes demonstrated that TR100 had minimal impact on contractile function. In vivo data from the drug treated animals also showed no evidence of cardiac damage as measured by blood Troponin I levels and no changes in the intraventricular septum thickness of isolated hearts. These results demonstrate that it is possible to target distinct actin filament populations based on the tropomyosin composition. Next generation anti-Tm compounds with improved efficacy and specificity have now been developed. Preliminary data demonstrate that these compounds exhibit increased selectivity for transformed cells.

Conclusions: Taken together, our findings suggest that the anti-Tm compounds show a significant improvement in the therapeutic window compared to existing anti-actin agents. This novel approach and the development of the new class of anti-Tm compounds may be the key for disabling a long sought after target, the actin cytoskeleton, and may lead to a new class of chemotherapeutics active against a broad range of cancer types.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A178.