bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B16: Anti-angiogenic therapy using brivanib, a combined VEGF and FGF pathway inhibitor, in a mouse model of pancreatic neuroendocrine cancer (PNET), results in sustained vascular blockade, without evidence for evasive/acquired resistance in the form of VEGF-independent revascularization, in contrast to other VEGF inhibitors
Authors: Allen Elizabeth , Walters Ian B. , Rivera Ianne C. , Hanahan Douglas
Published: June 2012

Previous studies using a mouse model of PNET (RIP-Tag2) demonstrated that anti-angiogenic therapy using an antibody that specifically targets murine VEGFR-2, DC101, led to a transitory period of stable disease followed by VEGF-independent revascularization and relapse leading to tumor progression (Casanovas, et al. Cancer Cell. 2005; 8: 299–309). Profiling analysis indicated that FGF was strongly upregulated upon failure of anti- VEGF therapy. Addition of a recombinant adenovirus expressing an FGF-Trap near the end of the response phase impaired revascularization and slowed tumor growth, implicating the FGF family of ligands in evasion of VEGF-targeted anti-angiogenic therapy in this model of PNET.

Brivanib, a selective small molecule inhibitor of VEGFR-2,3 and FGFR-1,2,3 (Cai, et al. J Med Chem. 2008; 51:1976–80), is currently being evaluated in clinical trials in CRC and HCC. Brivanib's activity against FGFR signaling prompted investigation of its effects on evasive/adaptive resistance to selective VEGF inhibition. In the work to be presented, the efficacy of brivanib has been characterized as a first line inhibitor versus two different VEGF inhibitors, DC101 and sorafenib, and as a second line inhibitor following the failure of the aforementioned compounds. DC101 is a pure VEGF inhibitor, while sorafenib is a multikinase RTK inhibitor of VEGFR-2, PBGFR , and RAF/MEK/ERK currently approved in renal and liver cancer, and in Phase 3 trials for multiple other indications.

Brivanib was comparable to FGF-trap as a second line inhibitor following DC101 failure, reducing tumor size and vascularity compared to continued DC101 monotherapy. Brivanib also showed reasonable efficacy in second line following sorafenib in short term trials. Notably, however, it could not reverse revascularization and tumor growth when sorafenib was allowed to fail in longer term trials. In contrast, first line brivanib therapy over comparable time courses that produced failure of both VEGF inhibitors resulted in tumor stasis and, in some cases, regression. Additionally, first-line brivanib therapy produced sustained blockade of angiogenesis, exhibited a trend for reduced invasiveness and metastasis in comparison to anti-VEGFR2 therapy alone, and significantly extended the lifespan of treated animals. Thus, while brivanib has clear benefits in second line therapy following failure of VEGF inhibitors, it may in some indications, as illustrated by this PNET model, be even more efficacious when used in a first line setting. The results from this mouse model of cancer may inform and facilitate design of future clinical trials of brivanib and other inhibitors of FGFR signaling.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B16.
Abstract B164: Role of endoplasmic reticulum stress in mechanism of action of antitumor quinols
Authors: Matthews Charles S. , McCarroll Andrew J. , Stevens Malcolm FG , Monks Anne , Bradshaw Tracey D.
Published: June 2012

The novel quinol PMX 290 is an experimental anti-tumor agent with potent activity against colon, renal and breast tumor cell lines in vitro (GI50 < 200 nM). PMX 290 disrupts thioredoxin/ thioredoxin reductase redox regulation and inhibits HIF-1 signaling. A pro-drug of PMX 290 is currently under development at Pharminox Ltd, UK. Here we present data suggesting an additional mechanism of action for PMX 290 highlighting a role for the induction of endoplasmic reticulum stress.

Treatment of HCT116 colon or TK-10 renal carcinoma cells with PMX 290 leads to the induction of a distinct vacuolar phenotype 4 – 6h after addition of the compound. These perinuclear vacuoles were identified as swollen endoplasmic reticulum (ER) as their membranes stain positively with a fluorescent ER dye. Furthermore when a fluorescent analogue of PMX 290 is added to cells it co-localized with the ER dye.

Microarray data from HCT116 cells treated with PMX 290 demonstrated the induction of a panel of mRNAs for genes involved in ER stress. These included GADD34, TRIB3, DNAJB9, BiP and CHOP as well as several genes upregulated by ATF4. Uncharacteristically for a quinol there was no increase in thioredoxin reductase mRNA levels although other markers of oxidative stress were upregulated (e.g. heme oxygenase 1).

The gene array results suggest that the PERK-eIF2alpha ER stress pathway is activated by PMX 290. Upon activation by ER stress PERK phosphorylates eIF2alpha which switches off general translation and allows the expression of the transcription factor ATF4. This switch to reduced protein synthesis and expression of ATF4 transcribed chaperone proteins lowers the load on ER and increases protein folding efficiency thus alleviating ER stress. A transient increase in eIF2aplha phosphorylation and a concurrent increase in BiP levels were detected byWestern blot in response to PMX 290 in HCT116 cells. In conclusion, ER stress appears to play an important role in the anti-tumor activity of PMX 290 and represents a novel mechanism of action for this class of compound.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B164.
Abstract B168: ANG1005: Results of a phase I study in patients with advanced solid tumors and metastatic brain cancer
Authors: Kurzrock Razelle , Gabrail Nash , Moulder Stacy , Noles Cherie , Smith Carrie , Guo Zhong , Bouchard Danielle , Churchill Wendy , Bento Paula , Neale Ann , Castaigne Jean-Paul , Sarantopoulos John
Published: June 2012

ANG1005 is a new generation taxane created from Angiochem's Engineered Peptide Compound (EPiC) platform. Studies have shown that ANG1005 gains entry into the brain compartment by targeting the low-density lipoprotein receptor-related protein (LRP) which is one of the most highly expressed receptors on the surface of the BBB. Once inside the brain, ANG1005 enters tumor cells using the same receptor-mediated pathway through LRP, which is upregulated in various cancer cells including metastatic brain cancer cells. Metastatic brain cancer is the most common intracranial neoplasm in adults with an estimated incidence of 200 000 cases each year in the United States alone. To date, treatment options are limited and marginally effective. A multi-center, phase I, open-label, sequential cohort, dose escalation study of ANG1005 in patients with advanced solid tumors and brain metastases is ongoing in the US. Study objectives include characterization of safety and tolerability and identification of maximum tolerated dose (MTD). ANG1005 is administered by IV infusion once every 21 days (1 treatment cycle) without premedication. After evaluating doses of 30–700 mg/m2, 650 mg/m2 was identified as MTD. Data including adverse events and hematological parameters indicate that ANG1005 is safe and well tolerated. The most common events occurring at a severity ≥ Grade 2 according to CTCAE, version 3.0 at MTD (n=20) were neutropenia (95% of patients; Grade 4 in 75%), leucopenia (100%; Grade 4 in 40%), anemia (55%; no cases of Grade 4), thrombocytopenia (40%; no cases of Grade 4) and mucositis (25%; no cases of Grade 4); these events have been transient and manageable with standard treatments. Neurocognitive data have shown that ANG1005 does not cause cognitive impairment and tests for antibodies demonstrate that ANG1005 does not elicit antibody production even in patients who have received up to 6 cycles of treatment. Pharmacokinetic data indicate linear ANG1005 bioavailability. Disease control (≥ stable disease) assessed by CT/MRI in this heavily pre-treated group of patients was achieved in 70% at doses > 300 mg/m2 including 7 patients who had previously progressed on taxane therapy. Partial response was observed in 30% of patients dosed > 300 mg/m2. Tumor types that showed the best improvements were breast, non-small cell lung and ovarian cancers. Furthermore, partial and complete responses were achieved in metastatic locations including the brain (67% disease control), liver (70% disease control), lung (67% disease control) and lymph nodes (80% disease control). Clinical data gathered to date indicate that ANG1005 shows promise as a potential treatment option for patients with advanced solid tumors and brain metastases.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B168.
Abstract B174: The effect of histone deacetylase inhibition on BRCA1 expression: A potential novel mechanism for enhancing platinum sensitivity in breast and ovarian cancer
Authors: Weberpals Johanne I. , O'Brien Anna , Garbuio Kyla , Clark-Knowles Katherine , Dimitroulakos Jim
Published: June 2012

Introduction: Targeting BRCA1 expression has been shown to sensitize Breast-Cancer 1 (BRCA1)-expressing tumor cells to platinum-based chemotherapies. The improved outcome of (BRCA1)-deficient breast and ovarian cancer is believed to be linked to the impaired ability to repair chemotherapy-induced DNA damage. However, therapeutically relevant agents that target BRCA1 expression have yet to be identified. In this study, we explore the effect of histone deacetylase inhibition (HDACi) on BRCA1 expression and platinum sensitivity in a breast and ovarian cancer cell line model.

Experimental procedures: BRCA1 mRNA and protein expression was determined by Q-PCR and Western blot, respectively. The efficacy of the HDACi, M344, to potentiate the cytotoxicity of platinum-based chemotherapeutics was evaluated in a range of breast and ovarian tumor cell lines using the MTT cell viability assay and confirmed by flow cytometry. The effect on DNA damage was measured by immunofluorescence staining and flow cytometry for H2A.X foci, a hallmark for the presence of DNA double strand breaks.

Results: Baseline BRCA1 mRNA and protein expression was variable in three breast (MCF7, T47D and HCC1937) and three ovarian (A2780s, A2780cp a cisplatin-resistant variant and OVCAR4) cancer cell lines. Expression of BRCA1 protein decreased in response to the addition of the HDACi, M344, in the four cell lines that express detectable levels of BRCA1 by Western blot analysis. BRCA1 mRNA levels decreased with the addition of this HDACi in all the breast and ovarian cancer lines evaluated. Treatment with M344 also increased the sensitivity of these tumor cells to cisplatin and carboplatin treatment in the cell lines with high BRCA1 expression. Furthermore, expression of BRCA1 protein and mRNA decreased in cells treated with the combination of HDACi and cisplatin. A2780s cells subjected to combination platinum and HDACi treatment demonstrated increased levels of DNA damage as assessed by the presence of phosphorylated H2A.X foci in comparison to either treatment alone. Conclusion: This study supports a novel mechanism of BRCA1 targeting using an HDACi resulting in enhanced platinum sensitivity in breast and ovarian cancer cells. Further studies are needed to determine if a HDACi/platinum combination has potential clinical relevance in a subset of breast and ovarian cancer patients.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B174.
Abstract B178: Nanoscale quantification of phosphorylated and unphosphorylated ERK and MEK isoforms differentiates tumor and nontumor clinical specimens
Authors: Fan Alice C. , Dermody Jessica L. , Kong Christina , Zhang Nancy , Orban Mathias W. , Pai Reetesh , Xu Liwen , Varasteh Ben , Tran Mai , Sridhar Kunju , Lee Lowen , Greenberg Peter L. , Felsher Dean W.
Published: June 2012

The MAP Kinase pathway is commonly dysregulated in cancer. We have developed the use of a highly sensitive microfluidic nano-immunoassay system (NIA) to perform detailed analysis of ERK and MEK activation in hematopoietic and solid tumors. We recently reported use of NIA for measurement of proteins in as little as 4 nanoliters of lysate from preclinical and clinical lymphoma and leukemia specimens (Fan et al, Nature Medicine 2009). Now, we present results measuring specific isoforms of MAP Kinase proteins in cells sampled from patients with solid tumors and Myelodysplastic Syndrome (MDS). First, we developed tissue collection, handling and processing protocols to produce reliable NIA results in a small number of cells obtained by minimally invasive techniques. We collected over 100 blood buffy coat and fine needle aspirate (FNA) specimens from normal controls and patients with MDS or solid tumors. Using a single antibody that recognizes both the phosphorylated and unphosphorylated isoforms of ERK, we can determine not only relative levels of phosphorylated isoforms of ERK protein, but also calculate the percent phosphorylation of ERK in each clinical specimen. We have developed a similar method to quantify MEK isoforms. NIA revealed that adenocarcinoma, squamous cell carcinoma, renal cell carcinoma, and MDS samples could be distinguished from non-tumor specimens based upon different patterns of phosphorylated ERK isoforms. To determine if signaling changes can be detected by NIA upon treatment of cells with novel targeted therapies, human leukemic TF1 cells were treated with a polo-1 kinase (PLK-1) inhibitor, ON01910 (Onconova, Inc.). We found the surprising result that only 5% of ERK1 and ERK2 were found to be phosphorylated in the TF1 cell line, whereas 45% and 90% of MEK1 and MEK2 were phosphorylated, respectively. Additionally, NIA analysis revealed a 20% decrease in the percent of phosphorylated MEK2 after treatment with ON01910. Taken together, our studies demonstrate that NIA can be used to identify specific changes in MAPK signaling pathway that distinguish normal from malignant cells as well as quantitatively measure the response of malignant cells to a targeted therapeutic.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B178.
Abstract B179: An improved method for array comparative genomic hybridization of formalin fixed paraffin embedded surgical pathology specimens
Authors: Suuriniemi Miia , Killian Keith , Walker Robert , Edelman Daniel , Meltzer Paul
Published: June 2012

Array comparative genomic hybridization (aCGH) has been widely used on high quality DNA obtained from fresh tissue samples to investigate chromosomal abnormalities associated with different cancers. Formalin fixed paraffin embedded (FFPE) tissue samples are an ideal source for retrospective and prospective genomic profiling studies because they are abundant in the archives and are the most common derivative of clinical surgical pathology analyses. However, DNA degradation and cross linking due to tissue processing and storage conditions has limited their prior utility for molecular testing. Here, we report an improved and reliable method to isolate, purify, and label DNA from FFPE tissue samples for high resolution aCGH.

Samples (n=67) were obtained from FFPE tissue blocks of >10yrs old and represented a variety of malignancies. Guided by hematoxylin and eosin stained sections, corresponding tissue block regions of maximal tumor cell density were targeted for extraction using a tissue microarray needle punch. Several changes to the Qiagen DNeasy kit, including buffers, temperatures, centrifugations, and elution conditions, were used to maximize DNA yield and produce a DNA derivative suitable for aCGH. Our method produced suitable DNA roughly 90% of the time, yet occasional impurities following Qiagen column purification persisted and manifested as a decreased 260/230 ratio, in which case we employed a simple additional column purification to render such samples usable. Non-enzymatic Universal Linkage System chemical labeling was used to directly couple fluorescent dyes with the sample and reference DNAs (1 ug each), which were then applied to an Agilent microarray. Data was analyzed in Nexus Copy Number software.

Average DNA yield was 1 ug per 0.6mm needle core. Our optional clean up step typically raised the 260/230 ratio from 1.5 to above 2. The majorities of samples (87%) were successfully labeled and produced good quality aCGH profiles with a median DLR score of 0.25 and signal-to-noise ratio of 17. We were also able to set up a threshold of the amount of label incorporation that reliably predicted the success of the aCGH. This will guide us in the future to test only samples expected to pass QC measures and thus not waste costly microarray components.

Our extraction and labeling method demonstrates the feasibility of successfully using DNA derived from FFPE tissue samples for aCGH, and brings this technique to the level of routine testing. Thus, our methodology removes many prior technical obstacles from aCGH based sample classification for diagnosis and clinical decision making using archival, current, and prospective FFPE specimens.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B179.
Abstract B193: Identification of selective inhibitors of ubiquitin specific proteases
Authors: Lopez Roman , Collura Vincent , Reverdy Céline , Planquette Cécile , Conrath Susan , Guedat Philippe , Delansorne Rémi , Daviet Laurent , Colland Frédéric
Published: June 2012

Regulated protein turnover is primarily controlled by the ubiquitin-proteasome system. The therapeutic efficacy of the proteasome inhibitor Bortezomib establishes this system as a valid anticancer therapeutic field. A promising alternative to targeting the proteasome itself lies upstream, at the level of the ubiquitin conjugation/deconjugation step to generate more specific anticancer agents. Ubiquitin-specific proteases (USP) are involved in the deubiquitination of specific target substrates regulating their stability, subcellular localization and/or activation status. USP represent a druggable target class due to their thiol-protease catalytic core which is amenable to pharmacological inhibition by small molecules. A genome-wide RNAi screen of the catalytically active human USPs in cancer-relevant cellular models and phenotypic assays allowed us to identify USP7/HAUSP and USP8/UBPY as promising cancer targets. Fluorescence-based screening assays using optimized USP substrates including various ubiquitin derivatives (ubiquitin precursor, branched ubiquitin chains) as well as specific, physiological substrates were developed. High-throughput screening performed on our chemically diverse library followed by different optimization programs resulted in the discovery of several series as novel USP inhibitors. Our progress made towards the specificity issue will be presented here with the identification of the first USP8- and USP7-specific series.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B193.
Abstract B202: A new curcumin analogue compound endowed with strong antitumor activity against neuroectoderma-derived cancers
Authors: Pisano Marina , Pagnan Gabriella , Dettori Maria Antonietta , Cossu Sara , Caffa Irene , Sassu Ilaria , Fabbri Davide , Emionite Laura , Cilli Michele , Pastorino Fabio , Palmieri Giuseppe , Delogu Giovanna , Ponzoni Mirco , Rozzo Carla
Published: June 2012

Sharing the common neuroectodermal origin, Malignant Melanoma (MM) and Neuroblastoma (NB) are tumors widely diffused among adult and children, respectively. Despite the current advances in treatment options, clinical prognosis of aggressive neuroectodermal cancers remains dismal. Chemotherapeutics currently in use are still unsatisfactory, therefore the search for novel therapies for such tumors is warranted. Curcumin, main component of curry spice, is one of the phytochemicals widely studied for its antioxidant, anti-inflammatory and anti-cancer properties. Moreover, it has been reported that curcumin exerts a good anti-proliferative activity on neuroectodermal tumor cells by inducing apoptosis. Recently, we have synthetized and tested in vitro various curcumin related compounds in order to select new antitumor agents displaying stronger and selective growth inhibition activity on both MM and NB cells. In this work, we have demonstrated that a new curcumin-related biphenyl compound, the α,β unsaturated keton (D6), was more effective in inhibiting tumor cells growth when compared to curcumin (D6 IC50 ∼1-2µM, vs curcumin IC50 ∼10-20µM). Normal fibroblasts proliferation was not affected by these treatments. Clonogenic assay, which measures the long-term effects of drugs on permanent cell growth arrest and cell death, showed a significant dose-dependent reduction in both MM and NB colony formation only after D6 treatment. Furthermore, TUNEL assay, Annexin V staining, caspases activation (such as caspases 3, 7, 9) and PARP cleavage unveiled the ability of D6 to cause tumor cell death by triggering apoptosis, similarly to curcumin, but with a stronger and quicker extent. Besides, these apoptotic features appear to be associated with loss of mitochondrial membrane potential and cytochrome c release, meaning that also the intrinsic apoptotic route took place. As reported in literature, curcumin inhibited NFkB nuclear translocation in neuroectodermal tumor cells and, therefore, D6 showed the same inhibitory effects but at lower doses. In vivo anti-tumor activity of curcumin and D6 was surveyed using sub-cutaneous MM and orthotopic NB xenograft models. In both experiments, D6 exibited significantly reduced tumor growth compared to both control and curcumin (MM: D6 vs control: P<0.001 and D6 vs curcumin P<0.01; NB: D6 vs both control and curcumin: P<0.001).

Our data indicate the biphenyl curcumin-related compound D6 could be considered as a good candidate to develop new adjuvant therapies against tumors with neuroectodermal origin.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B202.
Abstract B203: Mitochondria-targeted superoxide spin-traps for treatment of cancers with high oxidative stress
Authors: Basu Hirak S. , Vallerga Anne , Clark Paul A. , Kuo John S. , Joseph Joy , Kalyanaraman Balaraman
Published: June 2012

Many human malignancies such as prostate cancer (CaP) and primary brain tumor (GBM) exhibit high oxidative stress. Developing a successful therapeutic drug to treat advanced metastatic CaP and GBM is a major unmet medical need. It has been demonstrated that in certain chemo- and/or radiation resistant cancer cells with high oxidative stress, cells depend on reactive oxygen species (ROS) for survival and proliferation. Therefore, reducing ROS can be a successful strategy for developing chemotherapeutic drugs for use against chemo- and/or radiation-resistant tumors. The mitochondria are a major source of superoxide and other ROS. We focused on targeting spin-trapping nitroxide analogs at the mitochondrial interstitial space with appropriate linker length in order to remove the superoxide in order to reduce the cellular ROS levels without interfering with the mitochondrial electron transport chain. Our lead compound in this new class of drugs is Mito-tempol-C10. The drug successfully blocked ROS production in human CaP and GBM cell lines as determined by DCF dye oxidation assay. It exhibited marked growth inhibitory effect against androgen-dependent as well as androgen-independent cultured human CaP cells (IC50 < 1 µM) and similar growth inhibitory activity against several human GBM cells lines as well as cultured primary brain tumor stem cells as determined by DNA fluorescence measurement assay. Preliminary pharmacokinetic (PK) study show that the drug has serum half-life of approximately 45 minutes, but is retained by the tumor and other animal tissue for more than 24 hours. The drug is tolerated by ICR white mice, nude mice and NOD-SCID mice at a dose of 15 mg/kg i.p. given once every week for four weeks without any overt systemic toxicity. Preliminary efficacy study at this treatment condition showed marked effect on the growth of intracranially implanted, radiation-resistant U-87 MG human GBM cells in NOD-SCID mice and a small, but significant effect on the growth of androgen-independent PC-3 human prostate cancer cell xenografts growing on the flanks of nude mice. These results confirm that mitochondria-targeted spin-traps that reduce cellular ROS can be therapeutically effective in treatment of advanced, hard-to-treat human tumors with high oxidative stress.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B203.
Abstract B205: Evaluation of circulating CD34+ cells following treatment with anti-EGFL7 in preclinical models of efficacy
Authors: Huseni Mahrukh , Kadel Edward , Desai Rupal , Kan David , Cheng Jason , Yu Shang-Fan , Singh Mallika , Raja Rajiv , Ye Weilan , Hegde Priti
Published: June 2012

A significant existing challenge for anti-angiogenic therapy is that there are no validated biomarkers to either monitor dosing, response to therapy or escape from therapy. This limitation is a more significant challenge when the therapy in question targets a novel pathway with a unique MOA. Some generalized biomarkers have been identified to follow administration of anti-angiogenic therapy, and included in this class are circulating endothelial cells or CECs. CECs are a well-established marker of on-going and active angiogenesis1. In this report we describe the utility of measuring CECs in response to targeting a novel angiogenic protein, EGFL7. EGFL7 is an extracellular matrix protein that is highly expressed in endothelial cells of angiogenic vessels, including embryonic, tumor and injured vasculature. Inhibition of EGFL7 activity by anti-EGFL7 results in a reduction of tumor microvascular density and tumor growth in genetically engineered models of cancer such as RIP-T Ags. In order to identify a circulating biomarker for this response, we first explored the effect of anti-EGFL7 treatment on circulating CD34+ cells in preclinical tumor models, particularly in RIP-T Ag and MDA - MB-231. Analysis of circulating CD34+ cells showed a statistically significant decrease in circulating CD31−/CD34+/CD45dim cells in the blood of tumorbearing mice treated with anti-EGFL7. In order to characterize their molecular profiles, we isolated the two subpopulations of CD34+ cells - CD31−/CD34+/CD45dim and CD31+/CD34dim/CD45dim cells - from healthy adult human peripheral blood by flow cytometry. Subsequent microarray analysis of the two populations indicated that the gene expression profile of CD31+/CD34dim/CD45dim was indicative of terminally differentiated circulating endothelial cells (CEC), whereas the profile of CD31−/CD34+/CD45dim was suggestive of circulating progenitor cells (CPC.) Further evaluation by RT-PCR confirmed expression of EGFL7 in both CECs and CPCs. Additionally, we compared the prevalence of CD31+/CD34dim/CD45dim cells in whole blood from healthy adult and treatment naive NSCLC whole blood and confirmed that CD34+ cells could be used to follow administration of anti-EGFL7 in humans.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B205.
Abstract B223: Protein kinase C iota as a target for glioblastoma therapy
Authors: Lorimer Ian A. J. , Gray Douglas A. , Baldwin Mitchell
Published: June 2012

In spite of advances in surgery, radiation and chemotherapy, glioblastoma is still one of the deadliest forms of cancer. Two key features of the malignant nature of glioblastoma are its abnormal proliferation and its ability to invade both locally and to distant sites within the central nervous system. The phosphoinositide 3-kinase pathway is frequently activated by oncogenic mutations in glioblastoma, leading to activation of multiple downstream signaling molecules including protein kinase C iota (PKCι). Stable suppression of PKCι in glioblastoma cells with a short hairpin RNA caused a significant decrease in the proliferation of glioblastoma cells along with increased actin stress fiber formation and decreased cell motility and invasion. Live cell imaging was used to further assess the role of PKCι in glioblastoma cell motility and proliferation. While control glioblastoma cells form a coordinated leading edge lamellipodia and migrate substantial distances, cells stably depleted of PKCι show a loss of the ability to coordinate the formation of a functional leading edge lamellipodia and instead generate projections from all sides of the cell. These cells are therefore unable to move in a coordinated fashion. In addition live cell imaging showed that while glioblastoma cells round up and initiate mitosis, they are significantly impaired in their ability to complete mitosis. These effects on motility and mitosis were also seen when PKCι activity was inhibited with a myristoylated pseudosubstrate peptide. PKCι is therefore a promising new therapeutic target for glioblastoma.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B223.
Abstract B232: Preclinical evaluation of AMG479 a fully human insulin-like growth factor receptor-1 (IGFR1) antibody in ovarian cancer cells
Authors: Konecny Gottfried E. , Manivong Kanthinh , Guorong Ellen , Glas Ruth , Dering Judy , Anderson Lee , Ginther Charles , Velculescu Victor , Calzone Frank , Slamon Dennis J.
Published: June 2012

Background: Dysregulation of the insulin-like growth factor receptor-1 (IGFR1) signaling pathway has been implicated in the development and progression of ovarian cancer. Here we explore the therapeutic potential of AMG479 a fully human monoclonal antibody that targets the IGFR1 for the treatment of ovarian cancer.

Methods: We examined the effects of AMG479 on tumor cell growth in a panel of 40 characterized human ovarian cancer cell lines. Molecular markers for response prediction were studied using gene expression profiling, multiplexed phospho-protein ELISA, array comparative genomic hybridization, and mutational analyses. Associations between biomarkers and growth inhibition were analyzed using Spearman's rho correlation, and absolute levels were compared using the Mann-Whitney U test.

Results: Concentration-dependent antiproliferative effects of AMG479 were seen in nearly all ovarian cancer cell lines tested but varied significantly between individual cell lines. In vitro sensitive cell lines demonstrated significantly higher expression levels of IGFR1 (r = 0.36, p = 0.024), IGFBP5 (r =0.45, p =0.005), but lower levels of insulin receptor (r = -0.28, p =0.096). Eighteen of the 40 (45%) ovarian cancer cell lines demonstrated PI3K, KRAS, BRAF, or PTEN mutations. Cell lines without these mutations in the downstream signaling pathway of IGFR1 were significantly more sensitive to AMG479 when compared to those cell lines with downstream mutations (p = 0.026). Response to AMG479 was significantly correlated with its ability to inhibit (pre- versus post-treatment) phospho-p70S6K (r = 0.35, p = 0.028), and phospho-GSK-3 (r = 0.43, p = 0.005), but not with its ability to inhibit phospho-IGFR1 (r = 0.21, p = 0.183). Gene copy number gains or LOH of PTEN or PI3K, although common, were not associated with in vitro response to AMG479.

Conclusions: AMG 479 is a promising candidate for therapeutic use in IGFR1 dependent ovarian cancers. The present correlative study will help to identify biomarkers that may predict sensitivity to IGFR1 inhibition in ovarian cancer. PTEN, PI3K, or KRAS mutations may inversely correlate with sensitivity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B232.
Abstract B238: A phase 1 study of belinostat (PXD101) in combination with bortezomib in patients with advanced solid tumors or lymphoma
Authors: Nallapareddy Sujatha , Leong Stephen , Camidge Ross , Gore Lia , Diab Sami , Messersmith Wells , Lewis Karl , Weekes Colin , Gustafson Daniel , Jimeno Antonio , Zwiebel James , Espinoza-Delgado Igor , Eckhardt Gail , O'Bryant Cindy
Published: June 2012

Background: Belinostat (B) is a low molecular weight, synthetic hydroxamic acid derivative that inhibits class I and II histone deacetylases. Bortezomib (Bz) is a potent, selective inhibitor of the 26S proteasome. In preclinical studies, synergistic antiproliferative and pro-apoptotic effects were observed with the combination, providing the rationale for this study.

Methods: This phase 1 study is designed to determine the maximum tolerated dose (MTD) and to evaluate the safety and pharmacokinetic (PK) behavior of the combination of B and Bz. Each 21-day treatment cycle consists of B (600–1000 mg/m2) IV daily over 30 minutes on days 1–5 and Bz (0.7–1.5 mg/m2) IVP over 3–5 seconds on days 1, 4, 8, and 11 (days 2, 5, 8 and 11, cycle 1 only).

Results: To date, 26 patients have been enrolled (median age 59 [range 27–72]; median PS 1 [range 0–1]). Twenty-two patients were evaluable for toxicity and received a total of 58 treatment cycles (median 2 [range 1–6]). Four patients, 2 at dose level (DL) 3 (B: 600 mg/m2)/Bz: 1.3 mg/m2) and 2 at DL 5 (B: 1000 mg/m2)/Bz: 1.5 mg/m2) experienced a dose limiting toxicity (DLT). At DL 3, the DLTs were grade (gr) 3 dehydration and gr 4 thrombocytopenia. The exact relationship of the gr 3 dehydration and study combination is unclear as the patient had uncontrolled constipation and decreased oral intake as a result of a change in pain medications on cycle 1 day 1. This cohort was expanded to 9 pts to further determine tolerability of the study combination. No other DLTs were identified and dose escalation continued. At DL 5, DLTs included gr 4 thrombocytopenia and gr 4 fatigue. Most adverse events (AEs) have been mild to moderate. Gr 1–2 AEs (number of cycles) include anorexia (8), acute infusion reaction (5), fatigue (14), nausea (19), neutropenia (1), pain (12), phlebitis (6), thrombocyctopenia (11), and vomiting (12). Gr 3 AEs (occurring after course 1) include anorexia, dehydration, fatigue, nausea, vomiting, hypoalbuminemia, and elevation of alkaline phosphatase. Analysis of B PK demonstrates no statistical differences in the parameters between days 1 (B only) and 2 (B + Bz) for AUC, Cmax, clearance, or t1/2. Likewise, increasing Bz doses have no effect on B PK, whereas doses of B from 600 to 1000 mg/m2 result in dose-proportional increases in drug exposure. Four patients have maintained stable disease for 4–6 cycles of therapy.

Conclusions: Belinostat and bortezomib is a reasonable combination with a tolerable toxicity profile and no evidence of pharmacological interactions. Accrual is ongoing at the MTD, DL 4 (B: 1000 mg/m2)/Bz: 1.3 mg/m2). Additional biological assessments are planned in an expanded cohort of patients.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B238.
Abstract B240: Inhibition of MSP-RON signaling pathway in cancer cells by a novel soluble form of RON variant containing the complete sema sequences
Authors: Qi Ma , Zhang Kun , Wang Ming-hai
Published: June 2012

The RON receptor tyrosine kinase and its ligand macrophage stimulating protein (MSP) play a role in epithelial tumorigenesis. We report here a novel RON variant that antagonizes the RON-MSP pathway in various cancer cells. The variant was an 85 kDa soluble protein from a mRNA transcript with an insertion of 49 nucleotides between exons 5 and 6. The insertion created a stop code leading to a RON variant composed of a full 35kDa α-chain and a 45 kDa partial extracellular β-chain. The protein was featured by a sema domain, a hinge and a portion of the first IPT unit (designated as RON 85). RON 85 directly bound to the MSP, formed the MSP-RON 85 complex, and inhibited RON phosphorylation. RON 85 disrupted RON or RON 160 dimerization, prevented their phosphorylation, and attenuated their downstream signaling events. The action of RON 85 was specific to RON and had no effect on MET and EGFR. In colon and pancreatic cancer cells, RON 85 inhibited spontaneous or MSP-induced Erk1/2 and AKT phosphorylation, which results in impaired cell proliferation and colony formation. RON 85 also inhibited spontaneous or MSP-induced cell migration. We concluded that RON 85 is an antagonist to the MSP-RON pathway with therapeutic potentials for controlling tumorigenic phenotypes of cancer cells.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B240.
Abstract B241: Extended follow-up from a phase I study of AS1409, a novel antibody-cytokine fusion protein, suggests efficacy in malignant melanoma (MM)
Authors: Rudman Sarah M. , Jameson Michael B. , Savage Philip , Jodrell Duncan , Erlandsson Fredrik , Jones David , McKeage Mark , Spicer James F.
Published: June 2012

Introduction: AS1409 (huBC1-huIL-12) is a fusion protein comprising the humanized antibody, BC1, specific for the ED-B splice variant of fibronectin, and the human cytokine, IL-12. ED-B-fibronectin is expressed in tumor vasculature but has restricted distribution in normal tissue. IL-12 has immunostimulatory, anti-metastatic and anti-angiogenic properties. Responses to IL-12 occur in MM and RCC but with high systemic toxicity. AS1409 is designed to target IL-12 to tumor vasculature. We report extended follow-up from a phase I study to assess the tolerability, safety, pharmacokinetics and activity of AS1409 in patients with MM or renal cell carcinoma (RCC).

Methods: Patients with MM or RCC were treated in a dose-escalating trial of weekly i.v. AS1409 for 6 weeks at a starting dose of 15mcg/kg. Patients without unacceptable toxicity or disease progression could continue therapy. IFN-gamma and IP-10 were measured as markers of immune response.

Results: 13 patients (11 MM, 2 RCC) were treated (7 at 15mcg/kg, 6 at 25mcg/kg). DLTs observed at 25mcg/kg were transaminase elevation, fatigue and hemolytic anemia. Most common drug-related adverse events were Grade 1 or 2 and related to flu-like syndrome. Four patients continued to receive AS1409 beyond 6 weeks and one received 30 cycles of treatment. All patients showed elevation of IFN-gamma and IP-10 following the first dose. Tumor shrinkage was demonstrated in 5/9 evaluable MM patients - a partial response was confirmed in a patient with MM metastatic to lymph nodes and a further four patients achieved stable disease. One MM patient with previously rapidly progressive disease exhibited prolonged tumor shrinkage 12 months after discontinuing treatment. Anti-drug antibody (ADA) responses were seen in all patients. ADAs were shown not to neutralize the effect of AS1409 in vitro.

Conclusions: At 15mcg/kg, AS1409 was associated with manageable toxicity. Biomarker activation and objective radiological evidence of anticancer activity was observed at this dose in patients with MM, including one partial response and one patient with prolonged clinical benefit beyond 12 months from treatment. Further development of AS1409 in MM is warranted.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B241.
Abstract B244: Inhibition of metastases by a neutralizing TGFβ-specific antibody involves the activity of cytotoxic T lymphocytes and natural killer cells
Authors: Munroe Kenneth , Manning Charlene , Green Tessa , Tao Jingzang , Simon Kailene , McPherson John , Kaplan Johanne , Pinkas Jan , O'Brien Megan , Gregory Richard , Harper Jay , Lonning Scott
Published: June 2012

TGFβ plays a key role in metastasis by promoting epithelial to mesenchymal transition, up-regulating proteases that degrade extracellular matrix, enhancing tumor cell motility and suppressing antitumor immunity. Preclinical studies were conducted to determine the effect of neutralizing TGFβ on both primary tumor growth and metastases in in vivo models of experimental and spontaneous metastasis. Treatment with the pan-neutralizing TGFβ antibody, 1D11, as a single agent had limited effect on the growth of established, primary, syngeneic tumors (4T1 mammary carcinomas or B16-F10 melanomas) and did not enhance the efficacy of chemotherapeutic agents against these primary tumors. However, TGFβ antagonism was shown to have a significant effect on inhibiting metastasis to the bone following intracardiac injection of 4T1 cells. In addition, it was demonstrated that neutralization of TGFβ in B16-F10 melanoma models inhibited experimental pulmonary metastases following injection of cells into the tail vein, and inhibited spontaneous metastases from a subcutaneous primary tumor to the draining lymph node. Studies aimed at elucidating the mechanism of action of 1D11 in mice lacking specific immune effector cells suggest that the activity of cytotoxic T lymphocytes is important in the inhibition of metastases. The role of natural killer (NK) cells is less clear; NK cell activity is not required for inhibition of experimental metastasis to the lung, but is critical for the ability of 1D11 to inhibit spontaneous metastasis from a primary tumor to the draining lymph node. Investigation into the effects of neutralizing TGFβ on innate immunity and the tumor microenvironment are currently ongoing. Together these data suggest that neutralization of TGFβ significantly inhibits metastasis and anti-TGFβ strategies may be effective at treating metastatic disease in the clinic.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B244.
Abstract B245: The antitumor agent, Dp44mT, perturbs lysosomal function by binding copper and inducing oxidative damage
Authors: Lovejoy David B. , Jansson Patric J. , Brunk Ulf T. , Wong Jacky , Richardson Des R.
Published: June 2012

Current chemotherapies for solid tumors remain severely limited due to toxicity and the development of drug resistance. Hence, new agents are vital to explore in order to improve patient outcomes.

We have identified the novel chelator Dp44mT as a potent and selective anti-tumor agent that overcomes resistance to standard chemotherapeutics (Whitnall, M., Howard, J., Ponka P. and Richardson, D.R. PNAS USA 2006; 103:14901–6).

To understand its mechanism of action and potential anti-angiogenic activity, we have investigated its ability to interact with cellular copper (Cu). In contrast to its marked ability to potently mobilize cellular 59Fe, Dp44mT caused marked intracellular 64Cu accumulation. Cellular fractionation demonstrated that 64Cu was concentrated in a fraction containing lysosomes and mitochondria. Moreover, the lysosomotropic character of Dp44mT suggested that it may become concentrated in lysosomes. To test this, studies with the lysosomotropic agent, acridine orange, demonstrated that the Cu-Dp44mT complex potentiated loss of lysosomal integrity, indicating that the lysosome was a target of the compound.

This was confirmed using the fluorescent probe, pepstatin A BODIPY FL, which demonstrated relocalisation of the lysosomal enzyme cathepsin D from the lysosome to the cytosol after incubation with the Cu-Dp44mT complex. Further, the altered lysosomal membrane permeability was demonstrated by cleavage of the pro apoptotic Bcl2 family member, Bid. The Cu-Dp44mT complex was markedly more redox active than the Fe complex at lysosomal pH. This indicated that Cu mediated redox events were responsible for the cytotoxicity observed, in particular the induction of apoptosis induced by lysosomal damage. Further, the Cu chelator, tetrathiomolybdate potently attenuated Dp44mT mediated cell death.

Our results indicate the potent anti-tumor and anti-angiogenesis activity of Dp44mT is mediated not only by Fe chelation, but also via its binding of cellular Cu, leading to the generation of a cytotoxic complex.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B245.
Abstract B248: Characterization and preclinical development of LCI-1, a selective and potent Chk1 inhibitor in phase 1 clinical trials
Authors: Marshall Mark , Barda David , Barnard Darlene , Cox Karen , Diaz H. Bruce , King Constance , Nutter Suzanne , Westin Eric
Published: June 2012

Interference of DNA damage checkpoints has been demonstrated to be a highly effective means of increasing the cytotoxicity of a wide number of anti-cancer therapies. Ionizing radiation, DNA cross-linkers, topoisomerase inhibitors and anti-metabolites all cause severe cellular DNA damage and activation of multiple DNA damage checkpoints. Cell cycle arrest at these checkpoints protects injured cells from apoptotic cell death until the DNA damage can be repaired. In the absence of functioning DNA damage checkpoints, cells with damaged DNA proceed into premature mitosis followed by rapid apoptotic death. A key protein kinase involved in activating and maintaining the S and G2/M checkpoints is Chk1. Pharmacological inhibition of Chk1 in the absence of p53 leads to abrogation of the DNA damage checkpoints and has been shown to enhance the preclinical activity of many standard of care chemotherapeutic agents. LCI-1 is a novel small molecule inhibitor of Chk1 (IC50 = 7 nM). In cell-based experiments LCI-1 inhibited doxorubicin-dependent autophosphorylation of Chk1 (IC50 = 52 nM). Treatment of cells with LCI-1 alone caused a phenotype identical to that reported for cells depleted of Chk1, demonstrating the mechanism-dependent activity of the compound. Treated with 100 nM doxorubicin, HeLa cells arrested at the G2M checkpoint. When treated 24 hours later with LCI- 1, the arrested cells traversed the G2M checkpoint, allowing cells to proceed into mitosis with unresolved replicated chromosomes. Consistent with abrogation of the Chk1-dependent G2M checkpoint, HT-29 cells (mutant p53) treated with LCI-1 were more sensitive to killing by gemcitabine then were LCI-1 treated HCT116 cells (wild-type p53). In vivo, LCI-1 effectively inhibited Chk1 autophosphorylation, as well as released the S and G2M block induced by gemcitabine treatment. When used preclinically in combination with DNA damaging agents, LCI-1 increased DNA damage and cell death over gemcitabine alone. These results indicate that LCI-1 may prove to be an effective potentiator of DNA damaging therapies in the clinic. LCI-1 is currently undergoing clinical testing in combination with gemcitabine and pemetrexed.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B248.
Abstract B251: Combined anticancer effects of sphingosine kinase inhibitors and sorafenib
Authors: Beljanski Vladimir , Knaak Christian , Zhuang Yan , Smith Charles
Published: June 2012

Purpose: The pro-apoptotic lipid sphingosine is phosphorylated by sphingosine kinases 1 and 2 (SK1 and SK2) to generate the pro-survival lipid sphingosine-1-phosphate (S1P). We previously reported that inhibition of SK activity delays tumor growth in a mouse mammary adenocarcinoma tumor model. Because SK inhibitors and the multikinase inhibitor sorafenib both suppress the MAP kinase pathway, we hypothesized that the combination may provide enhanced tumor growth inhibition. Therefore, we evaluated the effects of two SK inhibitors, ABC294640 (a SK2-specific inhibitor) and ABC294735 (a SK1/SK2 inhibitor), alone and in combination with sorafenib on human pancreatic adenocarcinoma (Bxpc-3) and kidney carcinoma (A-498) cells in vitro and in vivo.

Experimental Design: Effects of the compounds as single agents or in combination in cell culture were assessed using the SRB assay, and CalcuSyn software was used to calculate synergistic or additive growth inhibition. Western blotting measured effects of compounds on signaling pathways. SCID mice bearing tumors of either Bxpc-3 or A-498 cells were treated with the agents and tumor growth was monitored.

Results: Exposure of either Bxpc-3 or A-498 cells to ABC29464/sorafenib or ABC294735/sorafenib combinations resulted in synergistic cytotoxicity, associated with increases in caspases 3/7 activation and apoptotic DNA fragmentation. In addition, strong decreases in ERK phosphorylation were observed in Bxpc-3 and A-498 cells exposed to the either sorafenib/ABC29464 or sorafenib/ABC294735 combinations. An increase in the phosphorylation of inhibitory site of c-Raf (S259) was also observed in the drug-treated cells. Oral administration of either ABC294640 or ABC294735 to mice led to a delay in tumor growth in both xenograft models without overt toxicity to the animals. Tumor growth delay was potentiated by co-administration of sorafenib.

Conclusions: These studies show that combination of an SK inhibitor with sorafenib causes synergistic inhibition of cell growth in vitro and potentiates antitumor activity in vivo. Thus, a foundation is established for clinical trials evaluating the efficacy of combining these signaling inhibitors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B251.