bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A108: A phase I and pharmalogical study of the broad-spectrum tyrosine kinase inhibitor JNJ-26483327, administered to subjects with advanced or refractory solid tumors
Authors: Konings Inge R. , de Jonge Maja J. , Burger Herman , van der Gaast Ate , van Bijsterveld Ludy EC , Winkler Hans , Hellemans Peter , Eskens Ferry A.
Published: June 2012

Background: JNJ-26483327 is a potent reversible multi-targeted tyrosine kinase inhibitor. Tyrosine kinases of Epidermal Growth Factor Receptor (EGFR), Her2, Her4, RET receptor, Vascular Epidermal Growth Factor Receptor (VEGFR)-3, and Src family (Lyn, Fyn, Yes) are inhibited at low nanomolar concentrations.

Methods: Objectives were to (1) determine safety, maximum tolerated dose (MTD) and dose-limiting toxicity (DLT), (2) characterize the pharmacokinetic (PK) profile, (3) explore preliminary evidence of antitumor activity, and (4) explore pharmacodynamic (PD) effects in skin biopsies and blood.

Results: 19 patients, 16 M/3F, median age 61 yrs (47-74) received JNJ-26483327 twice daily (BID) continuously at 100 mg (n=1), 200 mg (n=1), 400 mg (n=1), 800 mg (n=3), 1200 mg (n=3), 1500 mg (n=6) and 2100 mg (n=4). JNJ-26483327 was administered as oral solution (dose ≤ 1200 mg) or capsules (dose ≥ 1500 mg, 300 mg capsule). Skin biopsies were taken at days 0 and 28. At 2100 mg DLT consisted of a combination of grade 3 anorexia and fatigue in one patient, and due to substantial difficulty in intake in most patients, pill load at 2100 mg was dose limiting. JNJ-26483327 was well tolerated up to 1500 mg BID, with most common reported toxicities being nausea, vomiting, diarrhea, fatigue, skin rash. Plasma concentrations of JNJ-26483327 rapidly increased till about 3–4h post dose. PK showed significant inter-individual variability, but no marked deviation in dose-proportionality was observed up to 1500 mg BID. At 1500 mg BID, steady state plasma concentrations were 2–6 µg/l and AUCs 13–53 µg.h/ml and were in the active range as observed in xenograft mouse models. PD evaluation failed to show a substantial effect on expression of Ki-67, p27kip1, pMAPK, pAKT, and EGFR in paired skin biopsies. Serum levels of sVEGFR-2, VEGF-C and VEGF-D remained unchanged. No antitumor activity was observed in this study.

Conclusion: The recommended dose of JNJ-26483327 is 1500 mg BID. JNJ-26483327 shows a predictable pharmacokinetic profile. Additional PD research is planned to demonstrate biological activity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A108.
Abstract A126: Analysis of excision repair cross complementing group 1 (ERCC1) expression in squamous cell carcinoma of the head and neck (SCCHN)
Authors: Mehra Ranee , Cai Qi K. , Zhu Fang , Godwin Andrew , Burtness Barbara
Published: June 2012

Introduction: ERCC1 is a key enzyme in the nucleotide excision repair pathway and increased expression of ERCC1 is associated with cisplatin resistance in some cancers. Given the widespread use of platinum therapy for the treatment of SCCHN, we characterized the range of ERCC1 expression in SCCHN specimens using an immunofluorescence-based assay (automated quantitative analysis-tissue microarray [AQUA-TMA]). There is currently inadequate data regarding the range of ERCC1 expression in SCCHN, and how ERCC1 levels correlates with treatment outcomes, ERCC1 has been studied in SCCHN by standard immunohistochemistry, however, this method is limited by variability of techniques and subjectivity of interpretation, Therefore, there is a need for a highly quantitative technique which can be accomplished on formalin fixed paraffin embedded tissue and small samples, Our goal is to develop a reproducible assay that can eventually be incorporated into standard patient care practice.

Methods: Tissue microarrays containing head and neck squamous cell carcinoma samples obtained from patients treated at Fox Chase Cancer Center from 1999–2006 were constructed. Slides were stained by a modified indirect immunofluorescence method. Sections were incubated with ERCC1 antibody (8F1, Lab Vision) and wide-spectrum screening rabbit cytokeratin antibody (Dako Z0622). Prolong Gold mounting medium (P36931; Molecular Probes) containing 4,6-Diamidino-2-phenylindole (DAPI) was used to define tissue nuclei. A binary image (tumor mask) was created from the cytokeratin image of each histospot. ERCC1 levels were measured using fluorescent immunohistochemistry on the HistoRx PM-2000 image analysis platform and the data analyzed using AQUA algorithms. The in situ biomarker profiling system generated quantitative measurements of patient protein levels based on AQUA scores derived from fluorescent output of labeled markers. Both nuclear and non-nuclear (i.e., non-membrane/cytoplasmic) ERCC1 expression was measured

Results: 41 SCCHN specimens (24 oral cavity, 7 oropharynx, 5 glottic tumors, 3 unknown primary, 2 other) were analyzed. There were 25 males and 16 females and age at diagnosis ranged from 25 to 80 years (mean 64). The majority of patients were treated primarily by surgery. The range of AQUA scores was 33.66 to 1696.39 (mean 501.78, stnd deviation 284). The 25th percentile was 337.35, and the 75th percentile was 639.37. ERCC1 was predominantly nuclear (i.e., non-membrane/cytoplasmic) in the SCCHN tissue samples.

Conclusions: 1) It is feasible to determine a quantitative value of ERCC1 expression with AQUA. 2) There is a wide range of ERCC1 expression in SCCHN. 3) ERCC1 is predominantly localized to the nuclear compartment. 4) Ongoing work is being done to correlate ERCC1 levels with clinical features, sensitivity to platinum therapy, and survival.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A126.
Abstract A137: Chemotherapy molecule delivered by tumor-targeted salmonella enterica serovar typhimurium under the control of integrated modular gate
Authors: Yumei Dai , Forbes Neil S.
Published: June 2012

Several strictly or facultative anaerobic bacteria have attracted more and more attention in cancer therapeutics because of their targeting and accumulating abilities in solid tumors, and Salmonella enterica serovar Typhimurium is one of them. These bacteria were also adapted to deliver therapeutic factors and were applied alone or in combination with conventional therapeutics. The aim of this study is to test a new integrated gene expression system in S. Typhimurim SL1344, and the benefit of the new system in amplifying therapeutic molecule production during bacterium targeting and accumulating in solid tumor. We have constructed a synthetic genetic gate in S. Typhimurim SL1344 that integrates information from two promoters as inputs and actives a promoter output only when both input promoters are induced. The integration occurs via an interaction between L-arabinose concentration and bacterium cell density. When certain amount of L-arabinose were added in the system and bacterium population were higher enough, the tumor necrosis factor apoptosis inducing ligand(TRAIL) and fluorescent protein ZsGreen as the control were produced by bacteria. LS174T colon carcinoma cells were processed to form cylindroids between the bottom surface of a 96-well plate and the top surface of a set of polycarbonate cylindrical plugs attached to a polycarbonate lid to mimic solid tumor, and then cylindroid cultures were inoculated with S. typhimurium SL1344 carrying TRAIL or ZsGreen expression system. 0.2% (w/v) L-arabinose was added into the well after 12hr inoculation, and then washed out after 2hr induced. Extents of apoptosis in cylindroids were tested by caspase-3 activity assay. Results showed that the bacteria accumulated inside cylindroids indicated by ZsGreen expression, and the average normalized intensity of apoptotic cell was higher in the cylindroids inoculated by the bacteria carrying the TRAIL than those inoculated by the bacteria carrying the ZsGreen. It can be concluded that the constructed integrated modular gate could effectively control bacteria to produce functional chemotherapy molecule in solid tumor at desirable time and places.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A137.
Abstract A138: Development of potent inhibitors of the DNA-dependent protein kinase (DNA-PK)
Authors: Cano Celine , Curtin Nicola , Hardcastle Ian , Golding Bernard , Smith Graeme , Newell Herbie , Griffin Roger
Published: June 2012

The cellular response to DNA double-strand break (DSB) formation is an essential component of normal cell survival, following exposure to DNA-damaging chemicals and ionising radiation. The serine/threonine kinase DNA-dependent protein kinase (DNA-PK) is a member of the phosphatidylinositol (PI) 3-kinase related kinase (PIKK) family of enzymes, and plays an important role in DNA DSB repair via the non-homologous end-joining (NHEJ) pathway. DNA-PK inhibitors may, therefore, be useful as agents to improve the activity of radio- and chemo-therapy in the treatment of cancer.

Identification of the lead benzo[h]chromen-4-one DNA-PK inhibitor NU7026 (IC50 = 0.23 uM) guided the development of the potent and selective ATP-competitive chromenone NU7441 (DNA-PK IC50 = 30 nM). Structure-activity relationship studies for DNA-PK inhibition by chromenone-derivatives were conducted in conjunction with homology modelling. Library synthesis was undertaken employing a solution multiple-parallel approach, by O-alkylation or N-acylation of the appropriately substituted NU7441 derivatives, respectively, followed by reaction with a range of amines to afford the target compounds. These studies resulted in the identification of KU-0060648, which combines high potency as a DNA-PK inhibitor (IC50 = 8.6 nM) with good aqueous solubility as an acid salt. The further development of KU-0060648 and analogues will be described.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A138.
Abstract A14: sFLT01: An antiangiogenic protein that neutralizes placental growth factor
Authors: Bagley Rebecca G. , Kurtzberg Leslie , Weber William , Nguyen Tri-Hung , Lovewell Gwen , Nguyen Cokey , Yao Min , Roth Stephanie , Krumbholz Roy , Crawford Jennifer , Pechan Peter , Shankara Srinivas , Scaria Abraham , Schmid Steve , Brondyk Bill , Kaplan Johanne , Teicher Beverly A.
Published: June 2012

Introduction: Anti-angiogenic agents have shown clinical value in combination with chemotherapy by targeting the VEGF pathways. The development of tumor vasculature in human renal cell carcinomas (RCC) is stimulated not only by VEGF but by other angiogenic growth factors such as placental growth factor (PlGF) [1,2]. In the current study, we used a novel fusion protein, sFLT01, that includes human VEGFR1 domain 2 and an IgG1 Fc, which binds to both human VEGF and PlGF. The anti-angiogenic activity of sFLT01 was evaluated in several preclinical models of RCC.

Methods: The binding affinity of sFLT01 for PlGF was evaluated in vitro in binding assays quantified by ELISA and Octet and in proliferation assays where endothelial cells are stimulated by PlGF. The ability of sFLT01 to slow tumor growth was evaluated in the 786-O human RCC xenograft model as well as in the mouse RENCA RCC orthotopic and subcutaneous tumor models. sFLT01 was delivered by intraperitoneal injection twice per week at doses ranging from 5–25 mg/kg. Microvessel density (MVD) and other vascular parameters were analyzed in the syngeneic and xenograft tumors by immunohistochemical methods with an antibody against mouse CD31 to identify endothelial cells.

Results: sFLT01 bound to human PlGF with great affinity and inhibited HUVEC proliferation with an IC90 of approximately 2.3 nM. Subcutaneous 786-O RCC tumors were inhibited at a dose of 25 mg/kg and median survival time was increased by 33% in the orthotopic RENCA model. sFLT01 reduced MVD in subcutaneous RENCA tumors by approximately 50% and lumen area by approximately 66%. In the 786-O xenograft model, 5 or 25 mg/kg sFLT01 reduced the blood vessel area, lumen size, and vessel perimeter compared to control.

Conclusions: sFLT01 is effective at inhibiting blood vessel growth in tumors by binding VEGF and PlGF thereby preventing the development of new vasculature and decreasing existing vasculature. sFLT01 reduces intratumoral blood vessel count, prolongs survival, and delays tumor progression in the RENCA and 786-O renal cell carcinoma models. As an anti-angiogenic agent, sFLT01 may be useful in treating human renal cell carcinomas.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A14.
Abstract A140: Identification of substituted isoindolinones as potent inhibitors of the MDM2-p53 protein-protein interaction
Authors: Watson Anna , Liu Junfeng , Armstrong Elizabeth , Blackburn Timothy , Endicott Jane A. , Golding Bernard T. , Griffin Roger J. , Lu Xiaohong , Newell David R. , Noble Martin E.M. , Riedinger Christiane , Lunec John , Hardcastle Ian R.
Published: June 2012

The p53 tumor suppressor plays a pivotal role in the cell by reacting to stress, which may be caused by hypoxia, DNA damage, or oncogenic signalling. Activation of p53 protein results in the transcription of a number of genes that govern progression through the cell cycle, the initiation of DNA repair, and apoptosis. The activity of p53 is tightly regulated by the MDM2 protein, which is transcribed in response to p53 activation. MDM2 binds to and inactivates p53 and also ubiquitylates the MDM2-p53 complex to target it for proteosomal degradation. In normal cells the balance between active p53 and inactive MDM2-bound p53 is maintained in a negative feedback loop.

Inhibition of the MDM2-p53 protein-protein complex by small molecule inhibitors is expected to reactivate normal p53 pathways in cells overexpressing MDM2, consequently exerting an anti-cancer effect. Potent small molecule inhibitors of the MDM2-p53 interaction have been identified e.g. the Nutlins [Science 2004, 303, 844], the benzodiazepinediones and indolinones [J. Med. Chem. 2006, 49, 3759–3762]. The antitumor activities of these compounds in cellular and in vivo models are promising.

We have reported previously inhibitors of the MDM2-p53 interaction, based on an isoindolinone scaffold [J. Med. Chem. 2006, 49, 6209–6221], and SAR studies leading to compounds with improved potency e.g. NU8345A; IC50 = 171 ± 15 nM. NMR structural studies suggested that substitution of the isoindolinone A-ring may provide additional favourable interactions with the protein. A small series of examples was prepared, and 4-substitution with small hydrophobic groups was found to improve potency e.g. rac-4-methyl-3-(4-chlorophenyl)-3-((1-(hydroxymethyl) cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (NU8405; IC50 = 274 ± 35 nM) and rac-4-chloro-3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (NU8406; IC50 = 143 ± 26 nM). Introduction of a 6-tert-butyl group was also found to be favourable i.e. rac-6-tert-butyl-3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (NU8399; IC50 = 152 ± 27 nM). Resolution of the enantiomers of NU8406, using chiral HPLC, gave NU8406A and NU8406B (IC50s of 43.8 ± 6.2 nM and 1.27 ± 0.08 M, respectively).

The cellular activities of key compounds have been determined and show dose dependent induction of p53 regulated genes by Western blotting. NU8406 shows selective cytotoxicity in p53wt HCT116 cells compared with the HCT116−/− line (GI50s = 9.8 ± 1.1 and 21.3 ± 3.1 M, respectively) which is comparable to Nutlin-3 (ELISA IC50 = 66.2 ± 5.7 nM; GI50s = 8.9 ± 0.5 and 38.8 ± 3.4 M, respectively).

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A140.
Abstract A143: Novel temozolomide analog may overcome MGMT and MMR
Authors: Zhang Jihong , Stevens Malcolm FG , Kerschmeier Paul , Hummersone Marc , Laughton Charles A. , Bradshaw Tracey D.
Published: June 2012

Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer. The current standard of care for GBM patients includes surgery, radiotherapy and oral alkylating agent temozolomide (TMZ) chemotherapy. The cytotoxic activity of TMZ is dependent on DNA repair pathways, being inversely related to O6-methylguanine methyltransferase (MGMT) levels and dependent on functional mismatch repair (MMR). Intrinsic or emergent acquired resistance to TMZ presents severe obstacles to successful treatment. Therefore, TMZ analogs have been designed to overcome TMZ resistance, potentially possessing a broader spectrum of anti-cancer activity. Activities of TMZ and novel analogs were tested against 2 isogenic glioma cell line pairs: SNB19V and U373V (vector control cell lines, low MGMT) and SNB19M and U373M (MGMT overexpressing counterparts). TMZ and the corresponding triazene MTIC demonstrated 13.2- and 9.3-fold resistance in SNB19M cells; 5.4- and 4.25-fold resistance in U373M cells (compared to each vector transfected isogenic pair). Analog DC-010-116, where methoxycarbonylmethylene (CH2CO2CH3) replaces the methyl group of TMZ, and its corresponding triazene exerted growth inhibitory activity in TMZ sensitive (V) and TMZ resistant (M) cells, with GI50 values < 50 µM in all 4 cell lines. In addition, MMR deficient HCT 116 colon carcinoma cells, resistant to TMZ (GI50 590.6 µM) responded to DC-010-116 treatment (GI50 44.6 µM). Therefore, DC-010-116 exerts anticancer activity irrespective of MGMT and MMR status in glioma and colon carcinoma cells. Moreover, cross-resistance to DC-010-116 was not observed in glioma cell lines with acquired TMZ resistance (SNB19VR; U373VR). DC-010-116 blocked SNB19V, SNB19M and SNB19VR cells in G2/M and caused apoptosis in a time and dose dependent manner. Cellular DNA damage was detected by Alkaline Comet Assay, and H2AX phosphorylation indicated formation of lethal DNA double strand breaks following DC-010-116 treatment. DC-010-116 showed 3.7- fold difference in activity in paired base excision repair (BER) deficient/proficient CHO cell lines; furthermore, PARP inhibition potentiated sensitivity of HCT 116 cells to DC-010-116 (3-fold). However, DC-010-116, a likely substrate for plasma esterase activity, was devoid of activity in vivo. Accordingly the hydrolyzed carboxylic acid product (CH2CO2H) was inactive in vitro. In conclusion, we demonstrate that the anticancer activity of DC-010-116 escapes MGMT and MMR, blocks glioma cells in G2/M and induces apoptosis. DNA damage, recruiting BER, was detected. However, in vivo activity was compromized by plasma esterase activity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A143.
Abstract A151: Differential mechanisms of acquired resistance to IGF-1R antibody therapy and a small molecule inhibitor, BMS-754807 in a human rhabdomyosarcoma Rh41 model
Authors: Carboni Joan M.
Published: June 2012

Background: Overexpression and/or overactivation of IGF-1R have been linked to a number of cancer types including, colon, breast, prostate, lung and sarcoma. These findings support IGF-1R as a promising therapeutic target. Agents targeting IGF-1R, including IGF-1R antibodies and small molecule inhibitors are currently in clinical development. A common phenomenon resulting in clinical failures is the development of resistance to anti-cancer therapies. In the present study, the goal is to compare the mechanisms of acquired resistance to mAb391, an IGF-1R-blocking antibody and to BMS-754807, a small molecule dual inhibitor targeting IGF-1R/IR, and to provide insights on biomarkers for early detection of resistance and differentiation of IGF-1R targeted therapies.

Methods: The human Rhabdomyosarcoma cell line Rh41, which is sensitive to IGF-1R inhibition and responsive to both agents, was used to develop two acquired-resistant cell lines, Rh41-807R and Rh41-mAb391R. In addition, tumor xenograft models were developed from the resistant cell lines. Gene expression profiling was performed on the sensitive parental (Rh41), Rh41-807R, and Rh41-mAb391R cells and tumors to identify biomarkers involved in acquired resistance to BMS-754807 and/or mAb391. RNA expression profiles for selected biomarkers from both cells and tumors correlated well with protein expression patterns, as determined by Western blot analysis.

Results: Differences in resistance mechanisms between Rh41-807 and Rh41-mAb391 resistant lines were observed and each utilizes different alternative growth signaling pathways as an escape mechanism. PDGFR was found to be overexpressed at the RNA level in both Rh41-807R cells (89-fold) and tumors (27-fold) and was constitutively activated; siRNA knockdown of PDGFR in Rh41-807R re-sensitized the cells to BMS-754807. Synergistic activities were observed when BMS-754807 was combined with agents that also inhibited PDGFR in the Rh41-807R models. AXL RNA expression was found to be elevated in Rh41-mAb391R (8-fold), but down-regulated in Rh41-807R cells (5-fold). In addition, BMS-754807 was active in Rh41- mAb391R cells and able to overcome resistance to the IGF-1R antibody treated cells. However, the converse was not true i.e., mAb391 did not overcome resistance in the Rh41-807R cell line.

Conclusions: PDGFR overexpression and activation may drive resistance to BMS-754807, whereas differential resistance mechanisms are involved in resistance to the IGF-1R antibody. The activity of BMS-754807 in the Rh41- mAb391R acquired-resistant model suggests that treatment with BMS- 754807 may overcome resistance in patients who have failed treatment with IGF-1R antibodies.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A151.
Abstract A167: Simvastatin is synthetically lethal to the loss of LKB1 in lung cancer cells
Authors: Helbling Marianne M. , Schmitt Iris , Greiner Erich , Krek Wilhelm
Published: June 2012

‘Synthetic lethality’ offers an attractive therapeutic strategy for the development of cancer-specific cytotoxic agents. We therefore decided to exploit the genetic differences that exist between the tumor cell and the normal cell by searching for drugs that display ‘synthetic lethal’ interactions with the cancer-associated LKB1 mutation that occurs in about 30% of human lung adenocarcinomas.

Two genes are said to be ‘synthetic lethal’, if disruption of either individual gene is compatible with viability, but if loss of both causes death. It follows that a drug that is synthetic lethal to LKB1 should kill only the mutated LKB1 negative cells and spare normal cells.

A cell-based high-content screen for cell death was carried out with 48,000 small molecule compounds on an isogenic cell line pair that differs in its status of LKB1 (wild type versus mutant). We identified Simvastatin, an inhibitor of HMG-CoA reductase and widely used cholesterol-lowering drug, as one of the main hits. Other statins in the screening set, such as Lovastatin and Compactin, displayed similar activity.

Follow-up experiments confirmed synthetic lethal interactions between Simvastatin and LKB1-deficiency. Importantly, addition of mevalonate, the product of the HMG-CoA reduction rescued and siRNAs targeting HMG-CoA reductase further enhanced cell death induced by Simvastatin in LKB1-negative cells. Furthermore, Simvastatin specifically inhibited LKB1-negative tumor growth in a xenograft model in nude mice. The synthetic lethal relationship between Simvastatin and LKB1-deficiency indicates statins as potential cancer therapeutics for LKB1-negative tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A167.
Abstract A171: Cytotoxicity of novel substituted purine derivatives against liver cancer cells
Authors: Guven Ebru Bilget , Tuncbilek Meral , Ersahin Tulin , Demir Zerrin , Ozturk Mehmet , Atalay Rengul Cetin
Published: June 2012

Cytotoxic purine and purine nucleoside analogs (PNA) are widely used in cancer treatment (1). In this study, three newly synthesized substituted purine derivatives, AUM18, AUM23 and AUM32* were evaluated for their cytotoxicities by Sulforhodamine B (SRB) assay initially on Hct116 (colon), T47D (breast), and Huh7 (liver) human carcinoma cell lines (2). The IC50 values for the cell lines analyzed were ∼2.0µM for AUM18, ∼0.5µM for AUM23 and ∼5nM for AUM32. The growth inhibitory effects that we observed with these PNAs were comparable to those of the known anticancer agents Camptothecin (IC50 ∼ 10 nM) and 5-Fluorouracil (IC50 ∼10µM). Subsequently, the cytotoxicity of each molecule was tested on the HCC cell line panel composed of Huh7, HepG2, Hep3B, Mahlavu, PLC, SkHep1, Focus, SNU-182 and SNU-475 cells. The IC50 values were again in micromolar varying between 0.1 to 10 µM range. However, we did not observe significant cytotoxic effect on SNU-182 cells with AUM18 PNA and on SNU-475 with AUM32. The differential inhibition that we observe, can be considered as an indication of the specificity of these inhibitors against their target. They might interfere with the activity of certain kinases rather than acting as a multi-kinase inhibitor. Furthermore, we evaluated the protein kinase activity inhibition potential of these novel purine analogs. Therefore, the kinase assay based on the detection of the amount of ATP in the reaction mixture through bioluminescence was performed with Huh7 and Mahlavu cells. Cells incubated with these novel kinase inhibitor candidates and Staurosporine (STS), a multi-kinase inhibitor used as positive control (4). The relative light units that we observed with novel PNAs were comparable to that of STS. Based on our observations, the newly synthesized PNAs, AUM18, AUM23 and AUM32 can be identified as protein kinase inhibitor candidates which must be further analyzed at the molecular level.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A171.
Abstract A172: Dicycloplatin in cancer
Authors: Yang Xuqing , Yu Jing Jie , Liang Xiaobing , Zhu Honglan , Mueller Michael D.
Published: June 2012

Dicycloplatin (DCP) is a new platinum-based drug developed in China. It possesses superior stability and water-solubility compared to cisplatin and carboplatin. Experimental findings in China demonstrate in vitro cytotoxicity of dicycloplatin against a variety of human cancer cell lines and significant in vivo antitumor activity in mouse tumor models. DCP toxicity profile of 210 mg/kg of LD50 compares favourably to 14.27 mg/kg for cisplatin and 164 mg/kg for carboplatin. Histoculture Drug Response Assay (HDRA) indicates clinical efficacy of chemotherapy for various cancer types including brain cancer. Pre-clinical and clinical studies in China suggest that DCP produces fewer side effects with a maximum tolerated dose of 650 mg/m2. In one case, DCP with radiotherapy successfully treated a lung cancer patient with brain metastasis. Polar surface area (PSA) and LogP calculations for estimating solubility and ability to cross the BBB indicate that DCP possesses the largest PSA (132) and CLogP (1), compared to carboplatin (106.2; −0.34) and cisplatin (53.6; −2.5). Investigations of molecular mechanisms at WVU suggest that DCP induces gene signature profile in human ovarian cancer cells through mechanisms similar to other platinum drugs.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A172.
Abstract A174: High-throughput screening for natural product inhibitors of human tyrosyl-DNA-phosphodiesterase (Tdp-1)
Authors: Bermingham Alun , Price Edmund , Marchand Christophe , Pommier Yves , Goncharova Katya , Evans Jason , McMahon James , O'Keefe Barry
Published: June 2012

Tyrosyl-DNA-phosphodiesterase I (Tdp-1) is part of the DNA repair complex that resolves irreversible topoisomerase I (Top1)-DNA cleavage complexes by catalyzing the hydrolysis of 3′-phosphotyrosyl bonds. A point mutation in the Tdp1 gene causes a neurological disorder called spinocerebellar ataxia with axonal neuropathy (SCAN1) (Takashima, 2002). SCAN1 cells display hypersensitivity to camptothecin (CPT), a potent Topoisomerase 1 inhibitor. Additionally, overexpression of a human Tdp1 fusion protein alleviates some of the effects of CPT treatment (Barthelmes, 2004). These observations suggest that inhibitors of Tdp1 could act synergistically with CPT in a combined therapeutic treatment for certain cancers.

Despite attempts to identify synthetic small molecule inhibitors of Tdp-1, there are few verified inhibitors of this enzyme activity and none currently in clinical trials. While previous screening efforts have focused mainly upon compounds of synthetic origin (Antony, 2007), little effort has been directed towards the discovery of natural product derived inhibitors. The Molecular Targets Development Program (MTDP) at NCI-Frederick has developed a fluorescence-based assay for the rapid identification and characterization of natural product derived inhibitors of Tdp1 enzyme activity. Initial high-throughput screening work has identified several unique extracts showing potent inhibition of enzyme activity. Follow up work is currently underway to evaluate the molecular mechanism of isolated active natural compounds.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A174.
Abstract A188: Proapoptotic versus prosurvival function of the MAP kinase p38 induced by HDAC inhibitor in hematological malignant cells
Authors: Dupéré-Richer Daphné , Petruccelli Luca , Retrouvey Hélène , Dobocan Monica , Tomenson Miranda , Pettersson Filippa , Miller Wilson H.
Published: June 2012

Histone deacetylase inhibitors (HDACi) have recently emerged as promising anticancer agents. However, the mechanisms by which HDAC inhibitors arrest proliferation and induce apoptosis in tumor cells is far from clear. Activation of the stress MAP kinases and induction of DNA damage by different HDACi have been reported, however, the potential role of the MAP kinase p38 in the antitumor activity of these drugs has not been described. P38 is known to be activated independently or downstream of DNA damage. The purpose of this study was to elucidate the mechanisms by which the HDACi vorinostat (Zolinza®) triggers apoptosis in haematological malignant cells.

We show that DNA damage as well as activation of p38 occurs relatively early in acute myeloid leukemia (AML) cell lines after treatment with vorinostat. Using comet assays, we detected direct evidence of early DNA damage and western blotting revealed induction of the DNA damage response proteins ATM and Chk2. We performed cell cycle analysis, and observed within the vorinostat-treated AML cell population, cells exiting G1 and accumulating in the G2-M phase of the cell cycle, where they subsequently underwent apoptosis. Notably, downregulation of p38 by shRNA or inhibition of p38 and β activity by the inhibitor SB203580 significantly decreased both G2-M accumulation and apoptosis induced by vorinostat, indicating a pro-apoptotic p38 function.

Interestingly, several other HDACi tested all induced p38 activation but, depending on the HDACi, this activation was found to be either pro or antiapoptotic. The short-chain fatty acid sodium butyrate (NaB) requires p38 for induction of apoptosis, like vorinostat. On the other hand, LBH589, from the structural class encompassing vorinostat, does not depend on p38 for induction of apoptosis. Furthermore, p38 serves as a pro-survival signal when induced by the benzimide MGCD0103.

In conclusion, we have shown that vorinostat-induced apoptosis in AML cells is preceded by generation of DNA damage and accumulation of cells in the G2-M phase of the cell cycle. Further, G2-M arrest and apoptosis induction (but not DNA damage) by vorinostat requires activation of the p38 MAP kinase, which is not the case for all HDACi. Therefore, a better understanding of the role of p38 MAPK in the action of specific HDACi may help in the development of rational combination regimes including these targeted agents.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A188.
Abstract A189: Expression of fusion proteins in acute myeloid leukemia cells increases sensitivity to histone deacetylase inhibitors
Authors: Petruccelli Luca A. , Pettersson Filippa , Dupere-Richer Daphne , Rice Kim L. , Licht Jonathan D. , Miller Wilson H.
Published: June 2012

Acute Myeloid Leukemias (AMLs) are often characterized by chromosomal rearrangements that result in fusion proteins with aberrant transcriptional regulatory activities. These fusion proteins bind to gene promoters and recruit corepressors such as histone deacetylases (HDACs), which remodel chromatin into a closed conformation thereby silencing genes and contributing to a malignant phenotype. Aberrantly silenced genes include tumor suppressor and pro-differentiation genes. In addition, multiple groups have reported a DNA repair deficient phenotype concurrent with the expression of different fusion proteins in leukemia.

Small molecule HDAC inhibitors (HDACi) were devised as a strategy to reverse transcriptional repression. Indeed, many studies have demonstrated the ability of HDACi to re-sensitize leukemic cells to differentiating stimuli. However, other studies have revealed alternate methods by which HDACi exert anti-tumor activity, including induction of apoptosis that may be dependent on induction of ROS, MAPK signaling etc.

We find that low doses of HDACi, including Vorinostat and LBH589, induce cell death in the AML cell line U937 in a dose and time-dependant manner. Further, Vorinostat induced cell death in U937 cells is preceded by DNA damage and a G2/M arrest. This correlates with reports that HDACi repress DNA repair, by down-regulating DNA repair genes and by acetylating repair proteins thereby impairing their repair function.

Due to the inhibitory effect of leukemic fusion proteins on DNA repair, we predicted that DNA damage induced by HDACi, and thus cell death, would be amplified in AML cells expressing PML-RARα and PLZF-RARα fusion proteins. Sensitivity to Vorinostat and LBH589 was tested in three U937 derived cell lines: PR9 (PML-RARα inducible), B412 (PLZF-RARα inducible) and SN4 (mock transfected control). Indeed, induction of PLZF-RARα increased sensitivity of B412 cells to both Vorinostat- and LBH-mediated cell death. Assaying for DNA damage using alkaline comet assay, PR9 and B412 cells displayed an increase in DNA damage when their respective fusion protein is expressed. Nonetheless, the contribution of DNA damage to HDACi-mediated cell death remains unclear and further study is necessary. These findings are significant as they point to fusion protein expressing AMLs as a target group that may respond better to HDACi-based therapies.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A189.
Abstract A216: Distinct peptides derived from the cochaperone Bag1-L inhibit prostate cancer cell growth
Authors: Maddalo Danilo , Neeb Antje , Jehle Katja , Shatkina Liubov , Cato Andrew
Published: June 2012

Introduction and objectives: Bag1 (Bcl-2 associated athanogene-1) is a family of proteins conserved throughout evolution, promoting cell survival and inhibiting apoptosis under stressful conditions. They are found overexpressed in several types of tumors and associated with poor prognosis. Intriguingly, the nuclear isoform of the Bag-1 proteins, Bag-1L, also exhibits pro-apoptotic properties through an unknown mechanism. In extensive deletion mutagenesis studies we identified sequences in the Bag-1L protein that exert pro-apoptotic action. The objective of the study is to characterize the mechanism of action of these sequences and to explore their use as candidate peptides for prostate cancer therapy.

Materials and Methods: In vitro inhibition of tumor cell growth was analyzed in clonogenic assay where prostate cells were transfected with plasmids expressing different Bag-1L peptides. In vivo growth inhibition was analysed by the injection of prostate cells stably expressing the Bag-1L peptides into the flank of athymic nude mice and analyzing their effect on tumor size in vivo. Histological analysis for apoptosis was carried out on the tumor samples. For analyzing the interaction partners of the Bag-1L peptides, the peptides were generated as fusion proteins with glutathione S transferase (GST), immobilized on glutathione agarose beads and allowed to interact with lysates from prostate cells. Specifically bound proteins were then eluted and analysed by mass spectrometry and by Western blotting.

Results: Overexpression of a 68 amino acid sequence of Bag-1L reduced the growth of 22Rv.1 prostate cancer cells but not of benign prostate hyperplasia cells in a clonogenic assay. Stable overexpression of this peptide in the 22Rv.1 prostate cancer cells also reduced cell growth in vivo in athymic nude mice. Histological analysis revealed massive apoptosis in the tumor cells expressing the peptide. Further deletion analysis of the 68 amino acid peptide showed that the N-terminal 40 amino acids but not the C-terminal 29 amino acids are sufficient for the growth inhibitory action. Protein-protein interaction studies using GST-pull down assay demonstrated that the growth inhibitory function of the peptides correlated with their ability to bind glucose regulated proteins 75 and 78 (GRP75 and GRP78). GRP75 and GRP78 have been implicated in tumor formation through their action in the mitochondria and in the endoplasmic reticulum in the regulation of the unfolded protein response.

Conclusions: We have therefore identified Bag1-L peptides which when expressed in prostate tumor cells reduce the growth in vitro as well as in a mouse model of tumor formation. The action of the peptides is to induce apoptosis and it is correlated with their ability to bind the molecular chaperones GRP75 and GRP78.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A216.
Abstract A245: Imidazo[2,1-b]thiazole and imidazo[1,2-a]pyridine amides as novel inhibitors of the insulin-like growth factor (IGF1R) and members of the epidermal growth factor family of tyrosine kinases
Authors: Fidanze Steve D. , Erickson Scott A. , Hubbard Robert D. , Wang Gary T. , Mantei Robert A. , BaMaung Nwe Y. , Clark Richard F. , Sorensen Bryan K. , Kovar Peter , Pappano William , Zhang Qian , Wilsbacher Julie L. , Hu Xiaoming , Tucker Lora A. , Cheng Min , Swinger Kerren K. , Stewart Kent D. , Wang Jieyi , Bell Randy L. , Davidsen Steven K. , Sheppard George S.
Published: June 2012

The insulin-like growth factor 1 receptor (IGF1R) and its ligands, IGF1 and IGF2 are associated with cell growth and resistance to apoptosis. The ErbB axis, consisting of 4 receptor tyrosine kinases (EGFR, ErbB2, ErbB3, and ErbB4) is a longstanding target of cancer chemotherapy. There is a growing body of literature suggesting that targeting a combination of the IGF and ErbB axes would be more effective than targeting either alone. A screen of Abbott's compound collection revealed a scaffold, the imidazothiazoles, which showed activity against EGFR. A similar scaffold from the literature had previously been reported to have IGF1R activity. Optimization of the imidazothiazoles, and a scaffold hop to imidazopyridines, has provided a series of compounds with amide head groups with kinase inhibitory activity against IGF1R, EGFR, and ErbB2. These compounds also demonstrated inhibition of p-IGF1R and p-EGFR in a human epidermoid carcinoma line (A-431), as well as inhibition of p-ErbB2 in a human gastric cancer cell line (N87). This poster will discuss the synthesis and optimization of the imidazothiazoles and imidazopyridines as multitargeted kinase inhibitors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A245.
Abstract A250: Preclinical combination efficacy and identification of biomarkers of MEK and PI3K inhibitors in phase I
Authors: Hoeflich Klaus , Merchant Mark , Den Otter Doug , Chan Jocelyn , Chan Emily , Orr Christine , Friedman Lori , Chan Iris , Prescott John , Berry Leanne , Koeppen Hartmut , Belvin Marcia
Published: June 2012

Introduction: The Raf/MEK/ERK and PI3K/Akt/mTOR pathways are two major effector pathways downstream of activated Ras and receptor tyrosine kinases. While Ras is an attractive cancer target, direct pharmacological targeting has so far been unsuccessful. We describe preclinical studies combining GDC-0973, an inhibitor of MEK, and GDC-0941, an inhibitor of PI3K, both of which are in Phase I trials.

Summary of Results: GDC-0973 is a selective allosteric orally bioavailable small molecule inhibitor of the MEK1/2 dual specificity kinases. It has nanomolar potency against MEK1/2 in cells, and has potency across a broad range of cancer cell lines, most notably in those that are mutated for B-Raf and K-Ras. GDC-0941 is a selective orally bioavailable small molecule inhibitor of Class I PI3 kinases that has been described previously. Given the widespread and frequently concurrent activation of the Ras/Raf/MEK and Ras/PI3K/Akt pathways in tumors such as melanoma, colorectal, non-small cell lung cancer, and pancreatic cancer, we carried out cellular and in vivo xenograft studies to evaluate the efficacy of GDC-0973 and GDC-0941, both individually and in combination. We demonstrate strong combination efficacy in B-raf or K-ras mutant melanoma, colon and NSCLC models, driven primarily by increased apoptosis. The combination resulted in modulation of markers of both the MEK and PI3K pathways, and we discovered several markers whose modulation correlated with the induction of apoptosis. Continuous suppression of the two pathways is not required for combination efficacy.

Conclusions: The preclinical data indicate that combined use of MEK and PI3K inhibitors can produce greater efficacy than either agent alone at tolerated doses. Additionally, biomarkers have been identified which correlate with induction of apoptosis in the combination regimens.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A250.
Abstract A258: Combined blockade of Src kinase and epidermal growth factor receptor synergistically inhibits growth of pancreas cancer
Authors: Nagaraj Nagathihalli S. , Herndon Jason , Merchant Nipun B.
Published: June 2012

The failure of conventional chemotherapeutic regimes to produce any meaningful impact on survival in patients with pancreatic cancer highlights a desperate need for novel treatment strategies. The purpose of this study was to translate the current understanding of activated tyrosine kinase signaling pathways in pancreas cancer into improved patient therapies by targeting two tyrosine kinases, the epidermal growth factor receptor (EGFR) and the non-receptor Src kinase, which act in complementary pathways. The biochemical effects of individual and combined drug treatment with dasatinib (Sprycel, BMS-354825), a potent Src kinase inhibitor, erlotinib (Tarceva, OSI-774), a tyrosine kinase inhibitor to EGFR and the nucleoside analog gemcitabine were examined in pancreas cancer cell lines in vitro and in vivo. IC50 for dasatinib (DTB), erlotinib and gemcitabine was determined by the MTT proliferation assay against 9 human pancreas cancer cell lines, and sensitive and resistant cell lines were identified. In vitro regulation of cell motility, proliferation, survival, migration, invasion and in vivo tumor growth inhibition was determined. Expression of Ki67, cleaved caspase 3, phosphorylated Src and AKT in tumor xenografts was determined following exposure to combination of drugs.

The combined inhibition of Src with DTB and EGFR with erlotinib results in cooperative attenuation of cell migration, invasion, proliferation and increases in apoptosis of pancreatic cancer cells. Combined inhibition of Src and EGFR also results in cooperative inhibition of multiple signaling pathways including FAK, AKT, Stat3, ERK, JNK, MAPK and decreases cyclin D1 at concentrations that are ineffective as individual agents. The addition of gemcitabine enhances the effects of combined inhibition of Src and EGFR compared to that seen with individual agents alone or in double combination. Mice with tumor xenografts of BxPC3 (sensitive cell line) and Panc1 (resistant cell line) cells treated with DTB, erlotinib or gemcitabine either individually or as two drug or three drug combinations showed that the most effective in vivo anti-tumor effects were seen with the triple combination of DTB, erlotinib and gemcitabine even in xenografts of resistant pancreatic cells.

Pharmacological inhibition of EGFR and Src, in addition to gemcitabine treatment, results in cooperative inhibition of pancreatic tumor cell growth and appears to overcome resistance of single and double agent therapy. These results show that activation of Src and EGFR signaling pathways is evident in pancreas cancer and may be a driver of poor prognosis and chemotherapy resistance and provide a rationale for combination therapy with erlotinib, DTB and gemcitabine in clinical trials of pancreas cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A258.
Abstract A40: MicroRNA expression patterns as potential biomarkers of responsiveness to the MEK1/2 inhibitor, AZD6244, in colorectal cancer cell lines
Authors: Spreafico Anna , Tan Aik Choon , Tentler John J. , Nallapareddy Sujatha , Pitts Todd M. , Eckhardt S. Gail
Published: June 2012

Background: miRNAs are small, ∼22 oligonucleotides which typically act as negative regulators of gene expression. Recent data have demonstrated that aberrant expression of miRNAs may play a key role in the development of certain cancers and may also be involved in resistance to anticancer therapies. In this study, we tested whether miRNA expression patterns in AZD6244-sensitive (S) or -resistant (R) CRC cell lines could be used as potential biomarkers of responsiveness to this agent.

Methods: Responsiveness to AZD6244 (ARRY-142886) was assessed by exposing 24 CRC cell lines to increasing concentrations of the drug and measuring proliferation using the sulforhodamine B method. Sensitivity and resistance to AZD6244 was defined as: S, IC50<0.8 M; and R, IC50 ≥0.8 µM. Baseline total RNA was isolated from 4 S and 3 R cell lines for miRNA and mRNA profiling on Dharmacon miRNA microarray and Affymetrix HG-U133 Plus 2.0 microarray platforms, respectively. For miRNA microarray data pre-processing, 342 miRNA probes with relative intensity of p≤0.01 in at least 2 of the 8 experiments were kept for further data analysis. For mRNA microarray analysis, probe sets representing the same gene were collapsed based on maximum values and the expression levels were converted to a rank-based matrix. Significance Analysis of Microarray (SAM) was used to determine the differentially expressed miRNAs in cell lines S or R to AZD6244.

Results: Out of 24 CRC cell lines analyzed, 7 S and 17 R lines were identified. Six miRNAs were over-expressed in the R cell lines with at least four-fold change vs. S and with a false-discovery rate (FDR)<0.01. TargetScan listed 1181 genes potentially regulated by these six miRNAs. miRNA-target and Affymetrix gene expression profiles were compared and 171 genes that correlated inversely with miRNA expression at FDR<5% were identified. To assess the pathways enriched by these target genes, we queried this gene list against the KEGG database. Interestingly, expression of genes involved in the MAPK signaling pathway were down regulated in the AZD6244 R cell lines, which correlated with overrepresentation of the miRNAs associated with these genes.

Conclusions: In this study, we determined that AZD6244 S and R CRC cell lines exhibit differential expression patterns of miRNAs, which may have potential utility as a predictive biomarker for sensitivity to this drug. Furthermore, we found that overexpression of specific miRNAs in AZD6244 R cell lines correlates with down regulation of genes involved in the MAPK pathway, which may confer resistance to AZD6244. Suppression of these candidate miRNAs may induce sensitivity to AZD6244 in resistant CRC cell lines and therefore may have clinical application in the targeted therapy of patients with advanced CRC.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A40.