bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C102: Reactive oxygen species-related killing of lung adenocarcinoma cells in culture and in vivo by diazeniumdiolate-based nitric oxide-releasing prodrugs
Authors: Maciag Anna E. , Chakrapani Harinath , Shami Paul J. , Saavedra Joseph E. , Anderson Lucy M. , Keefer Larry K.
Published: June 2012

Lung cancer is the most common and among the most deadly of human malignancies. Early detection is unusual and there are no completely effective therapies in most cases. Diazeniumdiolate-based nitric oxide (NO) - releasing prodrugs are a growing class of promising NO-based therapeutics. Here we show that JS-K (O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) and its homopiperazine analogue JS-36-25 are potent tumoristatic agents against lung adenocarcinoma cells both in vitro and as xenografts in mice. JS-K treatment led to 75% reduction of the growth of H1703 cells in vivo, and JS-36-25 appears to be even more effective, leading to 85% reduction of the tumors. We have shown previously that JS-36-25 has diminished reactivity towards glutathione. This drug property may translate to a prolonged half-life in blood, allowing selective accumulation of JS-36-25 in the tumor. This would explain its possibly higher in vivo efficacy compared to JS-K.

Differential lung cancer cells’ responsiveness to both prodrugs appears to be related to their endogenous levels of reactive oxygen species (ROS). The level of endogenous ROS correlated significantly with the drugs’ toxicity measured as IC50 values. The treatment of the lung adenocarcinoma cells with JS-K or JS-36-25 resulted in significant, 2-fold increase in ROS/RNS generation in the cells with high basal levels of ROS. The oxidative/nitrosative stress resulted in significant DNA strand break damage in less than 1 hour of incubation with 1 µM drug. Activation of SAPK/JNK stress pathway was observed within 30 minutes after the treatment was initiated. Initiation of apoptosis occurred early, after 1 hour of incubation with 1 µM compound, as indicated by FACS analysis, as well activation of caspases 3 and 7 and PARP cleavage. Taken together, the data suggest that diazeniumdiolate-based NO - releasing prodrugs may have application as a personalized therapy for lung cancer in patients whose tumors are characterized by high levels of ROS.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C102.
Abstract C110: Abstract B: Hedgehog pathway inhibition in young mice results in severe bone defects
Authors: Brechbiel Jillian L. , Ng Jessica MY , Curran Tom
Published: June 2012

Current treatment for the most common pediatric brain tumor, medulloblastoma (MB) compromises neurological and endocrine development, particularly in younger patients. Mutations in Patched (PTCH1), the receptor for Hedgehog (Hh) proteins, cause MB in approximately 30% of patients. Loss of PTCH1 leads to constitutive activation of Smoothened (SMO) and increased expression of downstream target genes such as GLI1. Previously, we investigated the use of the SMO inhibitor, HhAntag, in a mouse model of MB. Treatment of Ptch1+/−p53−/− mice with HhAntag completely eliminates spontaneous and transplanted tumors. However, treatment of young mice with HhAntag results in permanent defects in bone structure. Histological analyses of long bones from these mice showed a significant reduction in proliferating chondrocytes and an increase in the number of hypertrophic chondrocytes. In our current study, we have further characterized the bone toxicities caused by HhAntag treatment and are testing potential molecules that might lessen the severity of these toxicities. We treated P10-old mice for 4 or 6 days and analyzed their limbs at 0, 2, or 4 days after HhAntag treatment. We found that the severity of bone defects increases with the length of time the mice are treated with HhAntag. MicroCT and MRI analysis showed that exposure to HhAntag for as little as 4 days caused premature growth plate fusion and an increase in bone mineral density. In an effort to protect proliferating chondrocytes from the toxicities caused by HhAntag, we have identified several candidate molecules that have been shown to alter developing bone. One candidate, Parathyroid Hormone-Related Protein (PTHrP), is a downstream effector of the Hh pathway. Hh signaling activates the expression of PTHrP, which then signals to proliferating chondrocytes, preventing them from differentiating. Previous studies show that overexpression of PTHrP in developing bones delays chondrocyte hypertrophy. Therefore, exogenous PTHrP may offset the premature differentiation evident in HhAntag treated animals. We have identified a physiologically relevant dose of PTHrP that is sufficient to mediate a change in bone morphology. We are currently treating mice simultaneously with PTHrP and HhAntag, in an effort to reduce the HhAntag associated bone toxicities. Another candidate is the proteasome inhibitor, bortezomib. Bortezomib has been shown to alter Hh pathway activity and therefore may also reduce the toxicities associated with HhAntag treatment. We are currently establishing the proper dosing strategy appropriate for bortezomib treatment of young mice. We have shown that treatment of young mice with HhAntag leads to severe bone defects and are currently employing a multi-drug approach to lessen the severity of these defects.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C110.
Abstract C115: The KidsCancerKinome: Validation of Aurora kinases as potential drug targets in pediatric tumors
Authors: Westerhout Ellen M. , Kool Marcel , Molenaar Jan J. , Boer den Monique L. , Clifford Steve C. , Delattre Olivier , Geoerger Birgit , Lanvers Claudia , Pietsch Torsten , Serra Massimo , Shipley Janet , Vassal Gilles , Versteeg Rogier , Verschuur Arnauld C. , Caron Huib N.
Published: June 2012

The KCK consortium has validated Aurora kinase A and B as potential drug targets in six highly malignant pediatric tumor types (i.e Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, neuroblastoma, medulloblastoma and ALL). The stepwise procedure started with the extensive analyses of expression of human kinases using Affymetrix mRNA profiles of over 500 tumors and cell lines. Clustering analyses on the combined data of all tumor types revealed a cluster containing many G2M kinases that showed significantly higher expression patterns than in the reference tissues. Prominently present in the G2M cluster were Aurora kinase A and B, which expression could be correlated to poor prognosis in the individual tumor types in further analyses. Subsequently, lentiviral shRNA-mediated knockdown of AURKA and AURKB protein expression has been performed in cell line panels for each tumor type to evaluate the kinases for their potential as drug targets. Inhibition of the Aurora kinases resulted in significant phenotypes in several pediatric tumor cell lines ranging from growth inhibition to extensive cell death. The knockdown of AURKA or AURKB often leads to induction of apoptosis, although preceded by a different type of cell arrest. These findings were promising for further evaluation of Aurora kinase inhibitors in the core panel of sensitive pediatric tumor cell lines.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C115.
Abstract C125: Tanespimycin pharmacokinetics: Results of a randomized dose-escalation crossover phase 1 study of two formulations
Authors: Burris Howard A. , Jones Suzanne , Messina Marianne , Berman David
Published: June 2012

Introduction: Tanespimycin is an inhibitor of Hsp90, a chaperone for proteins involved in tumor growth and survival. In vitro, tanespimycin sensitizes tumors to anti-cancer therapies. Tanespimycin has demonstrated clinical activity as monotherapy and in combination with bortezomib in multiple myeloma pts and with trastuzumab in pts with HER2+ breast cancer.

Study Design: In this single-center, open-label dose-escalation study, pts were randomized to receive 1 dose IV of Cremophor-containing tanespimycin or tanespimycin suspension and then crossed over and received 1 dose of the other formulation. One cohort (3–6 pts) received 275 mg/m2 and 1 cohort (6–12 pts) received 340 mg/m2. After crossover, pts continued receiving tanespimycin suspension twice weekly on days 1, 4, 8, and 11 of each 21-d cycle until disease progression. Blood samples were collected on days 1 and 8 at the following times: predose, during infusion, 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h postdose. Plasma concentrations of tanespimycin and its metabolite 17-AG were quantified by LC/MS/MS. Investigators graded adverse events (AEs) using the CTCAE v3.0 and tumor responses using RECIST.

Results: A total of 17 pts were enrolled in the study: 5 pts in the 275 mg/m2 cohort; 12 pts in the 340 mg/m2 cohort. Fourteen pts continued treatment following crossover. The mean age was 59.5 y and 76.5% were women. The median time since diagnosis was 41.5 mos and most common primary diagnoses were breast cancer 29%, non-small cell lung cancer 12%, and larynx cancer 6%. The plasma concentration:time curves show biexponential decay with a rapid distribution followed by a slower elimination phase. Profiles for the Cremophor-containing and suspension formulations were similar with Cmax for the 275 mg/m2 dose of 8050 ng/mL and 5235 ng/mL, respectively, and Cmax for the 340 mg/m2 dose of 10,578 ng/mL and 9270 ng/mL, respectively. t1/2 ranged from 3–4 h for tanespimycin and 4–5 h for 17-AG. Tmax for all doses was approximately 1 h for tanespimycin and 1.5–2.6 h for 17-AG. The most common AEs (all grades) in the continuation phase were: vomiting (65%; Gr3/4 0%), diarrhea (53%; Gr3/4 18%), nausea (47%; Gr3/4 0%), fatigue (35%; Gr3/4 9%), and headache (24%; Gr3/4 0%). Of the 11 response evaluable pts, 7 pts had a best response of stable disease (duration ranged from 4.1+ to 35.6 wks).

Conclusion: Tanespimycin suspension was well tolerated in pts with solid tumors. Plasma concentration:time curves were similar for the 2 formulations with biexponential decay showing a rapid distribution followed by a slower elimination. These results support ongoing studies with twice weekly doses of 340 mg/m2 tanespimycin suspension.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C125.
Abstract C127: Factors affecting the pharmacokinetics (PK) and pharmacodynamics (PD) of PEGylated liposomal irinotecan (IHL-305) in patients with advanced solid tumors
Authors: Wu Huali , Infante Jeffrey R. , Jones Suzanne F. , Burris Howard A. , Chan Emily , Keedy Vicki L. , Bendell Johanna C. , Zamboni Beth A. , Kodaira Hiroshi , Ikeda Satoshi , Zamboni William C.
Published: June 2012

Background: Irinotecan (CPT-11) is a prodrug of SN-38 that has antitumor activity in a wide range of solid tumors. IHL-305 is a PEGylated-liposomal formulation of CPT-11. The PK of liposomal agents is highly complex due to the variability in the disposition of the inactive-encapsulated drug and the release of active-drug from the liposome. We previously reported that age, body weight, and monocytes affect the PK and PD of PEGylated liposomal agents. Thus, we evaluated the effect of these factors on the PK and PD of IHL-305 as part of a phase I study.

Methods: IHL-305 was administered IV over 1 h at 3.5, 7, 10.5, 14, 28, 33.5, 37, 50, 67, 80, 88, 120, 160, and 210 mg/m2 q 28 d. Serial plasma samples were obtained prior to infusion, at the end of infusion (EOI), and from 1 to 192 h after EOI. Plasma samples were processed to measure sum total (encapsulated + released) CPT-11, released CPT-11, SN-38 and SN-38 glucuronide (SN-38G). The total (lactone + hydroxy acid) forms were measured by HPLC. The area under the plasma concentration versus time curve (AUC) for each form was calculated from 0 to last sampling time and from 0 to ∞. The ratio of total body weight to ideal body weight (TBW/IBW) was calculated.

Results: 39 patients (pts) (13 male) were treated. Median (range) of age and TBW/IBW were 60 years old (yo) (42–75 yo) and 1.16 (0.81–2.7), respectively. There was high inter-patient variability in the PK disposition of sum total CPT-11, released CPT-11, SN-38 and SN-38G. Sum total CPT-11 is associated with linear and non-linear clearance. The ratio of SN-38 to released CPT-11 and SN-38G to SN-38 were similar to those reported after irinotecan. Pts whose age and TBW/IBW were greater than the median of the study had a 1.7- to 2.6-fold higher ratio of released CPT-11 AUC to sum total CPT-11 AUC. In pts < 60 yo and in pts ≥ 60 yo, the ratio of % decrease in monocytes at nadir to % decrease in ANC at nadir were 1.65 ± 1.36 and 1.24 ± 0.68, respectively. There was an inverse relationship between pts age and % decrease in monocytes at nadir with younger pts having a higher % decrease in monocytes. Pts with a higher % decrease in monocytes at nadir have an increased clearance of sum total CPT-11 and increased exposure of released CPT-11 and ratio of released CPT-11 AUC to sum total CPT-11 AUC.

Conclusions: PK and PD of IHL-305 are consistent with a PEGylated-liposomal carrier. IHL-305 has pharmacological advantages compared with irinotecan. The inter-patient variability in the PK and PD of IHL-305 was associated with age, body composition, and monocytes. Sponsored by Yakult Honsha Co., Ltd., Tokyo, Japan

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C127.
Abstract C13: Ascorbate induces cytotoxicity in prostate cancer cells through an autophagy pathway
Authors: Chen Ping , Chalmers Brian , Yang Jun , Li Benyi , Drisko Jeanne A. , Chen Qi
Published: June 2012

Ascorbate (Vitamin C) has been shown to act as a pro-oxidant agent under pharmacological concentrations, and thus can cause cancer cell death in vitro and inhibit tumor growth in animal models, through formation of H2O2. The cell death mechanisms induced by ascorbate need to be further elaborated.

The goal of our study was to determine whether ascorbate is cytotoxic to prostate cancer cells and if so, to determine the mechanism of ascorbate-induced cytotoxicity. We hypothesized that through generation of H2O2, pharmacological concentrations of ascorbate can deplete prostate cancer cell ATP and cause cell death through autophagy pathway. A panel of prostate cancer cell lines PC-3, LNCap, C4-2, LaPC4 and 22RV1 were incubated with millimolar range of ascorbate for two hours. Then ascorbate was washed out and cells were incubated in fresh media for 24 hrs before cell viability was assessed. Cell ATP levels were determined by HPLC. The results showed that 4 out of the 5 tested cell lines have IC50s < 5 mM, while the most resistant cell line LaPC4 has IC50 at 12 mM. Addition of catalase - a H2O2 scavenger to the culture media, completely protected the cells from the death caused by ascorbate, confirming the death is mediated by H2O2. PC-3 cell ATP levels significantly decreased after 2 hrs of incubation with 5 mM ascorbate, and did not recover over the following 24 hrs. To determine if ascorbate induced autophagy, western blot of PC-3 cells for LC3 was performed. The results showed an increase of LC3-II form at 2 hrs of ascorbate treatment, indicating the processing of LC3 protein to its lipidated form. At 24 hrs the LC3-II bands were decreased, which may due to degradation of LC3 proteins at the autophagosomes. We also generated cells (PC3-LC3G) that express a fusion protein of LC-3 and GFP. The GFP-LC3 protein punctation were observed at 4 hrs of ascorbate treatment, indicating a translocation of LC3 to the autophagic membranes. These results suggest that ascorbate induces cytotoxicity in prostate cancer cells through depletion of cell ATP and induction of autophagy. To further confirm this pathway, an autophagy inhibitor 3-MA is under testing, the degradation of GFP-LC3 is also under analysis to determine whether ascorbate increases the autophagic flux. Also, to confirm the role of ATP drop in ascorbate-induced autophagy, 3-aminibenzamide (3AB) and 4-hydroxyquinazoline (4HQ) are being used to prevent the drop in ATP level.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C13.
Abstract C134: Survivin suppressant YM155: A pharmacokinetic comparison between U.S. and Japanese populations
Authors: Cao Ying J. , Aoyama Yumiko , Kakihara Toru , Katashima Masataka , Sawamoto Taiji , Roy Michael , Wang Jimmy , Abeyratne Anura , Nakahara Takahito , Steinberg Joyce , Keating Anne
Published: June 2012

Survivin is a member of IAPs. YM155 is a compound that suppresses survivin, and has been shown to exhibit anti-tumor activity in preclinical and clinical studies. The current analysis is designed to compare the pharmacokinetics (PK) of YM155 between US and Japanese patient populations using data obtained from two phase 1 trials, one done in Japan and the other in the US. Patients with a confirmed solid tumor or NHL (US only) that were refractory to standard therapy or for which no standard therapy was available participated in these trials. YM155 was administered as a continuous IV infusion for 168 hours, every 21 days. During the first two treatment cycles, serial blood and urine samples were collected for up to 48 hours after the discontinuation of YM155 infusion on Day 7. Forty-one subjects in the US trial (including 5 subjects with NHL) and 33 subjects in the Japanese trial had adequate samples for PK evaluation. The maximum tolerated dose (MTD) was 4.8 mg/m2/day and 8.0 mg/m2/day in the US and Japanese populations, respectively. In both populations, median YM155 concentration appeared to reach steady-state 12–24 hours after the start of the YM155 infusion. After discontinuation of the YM155 infusion, plasma YM155 concentration declined rapidly in a biphasic pattern with mean terminal half-life between 6 and 29 hours. The geometric mean ratio [90 % confidence interval(CI)] of steady-state YM155 concentration (Css) and area under observed YM155 concentration-time curve extrapolated to infinity (AUCinf) between cycle 2 and cycle 1 was 1.09 (0.99 –1.29) and 1.02 (0.90–1.16) in the US, and 0.98 (0.80 – 1.21) and 1.00 (0.97–1.03) in the Japanese populations, respectively, suggesting that there was no time-dependence in YM155 exposure. The slope of regression of natural logarithm transformed Css and AUCinf on natural logarithm transformed dose (mean and 95% CI) was 0.66 (0.31–1.00) and 0.65 (0.29–1.00) in the US and 1.00 (0.86–1.14) and 0.99 (0.85–1.12) in the Japanese populations, respectively. At the US MTD dose of 4.8 mg/m2/day, Css (mean ± SD) during cycle 1 was 7.6 ± 2.6 ng/mL (n=24) in the US and 8.7 ± 1.4 ng/mL (n=6) in the Japanese populations. The geometric mean ratios (90 % CI) of AUCinf, Css, and clearance between Japanese and US populations were 1.06 (0.93 – 1.22), 1.14 (1.00 – 1.31) and 0.80 (0.70 – 0.92), respectively. In conclusion, data currently available suggests that PK of YM155 is time-independent in both the US and Japanese patient populations; there appear to be no differences between U.S. and Japanese patients in terms of blood plasma YM155 concentration and its time course, although high variability was observed.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C134.
Abstract C141: In vitro and in vivo characterization of two novel β-catenin RNA antagonists, EZN-3889 and 3892
Authors: Zhang Yixian , Sapra Puja , Kim Steven , Shi Victoria , Qu Zhengxing , Castaneda Stephen , Wang Maoliang , Zhu Peifang , Filpula David , Greenberger Lee M. , Horak Ivan D.
Published: June 2012

Background: β-Catenin is an important signaling molecule in the Wnt pathway that plays a key role in tumorgenesis. In the absence of Wnt signaling, the cytoplasmic level of β-catenin is kept low due to rapid proteasomal-mediated degradation of GSK3β phosphorylated β-catenin. Activation of Wnt signaling leads to the inactivation of GSK3β, resulting in stabilization and accumulation of β-catenin in the cytoplasm. Consequently, β-catenin translocates into the nucleus, where it binds with members of the T-cell factor (Tcf)/lymphocyte enhancer-binding factor family of transcription factors and activates the expression of many target genes important for cancer development. Most colon cancers have activating mutations in the APC tumor suppressor or in β-catenin itself. Furthermore, activating β-catenin mutations have been found in a variety of other tumors such as melanomas, hepatocellular carcinomas, skin, breast, and prostate cancer, whereas β-catenin is not activated in most normal tissues. Therefore, inhibition of β-catenin is likely to have therapeutic effects in many cancers. We report here the identification of two β-catenin LNA-based mRNA-antagonists, EZN-3889 and EZN-3892.

Material and Methods: In vitro, the ability of the compounds to inhibit mRNA, cell growth, and reporter gene were evaluated by qRT-PCR, MTS, and luciferase assays respectively, in multiple cell lines. In vivo, β-catenin mRNA down-modulation in liver and human tumors, which were grown on the flank of nude mice, was evaluated after intravenous administration of the compounds.

Results: These molecules were potent down-modulators of β-catenin (IC50 = 0.1 to 3 nM) as well as protein (> 80% at 20 nM) of multiple cell lines, when transfected into tumor cells. Growth inhibition appeared to correlate with the status of either APC mutation or activating β-catenin mutations, suggesting that β-catenin is the driver of cancer cells. Interestingly, we found that these two antagonists specifically down modulated β-catenin mRNA and protein effectively in multiple cell lines with a long-lasting effect in the absence of any tranfection reagent. In mice, both molecules significantly down modulated β-catenin mRNA in the liver.

Conclusions: β-catenin antagonists potently and specifically inhibited β-catenin mRNA expression both in vitro and in vivo after intravenous injection. Further studies will examine the antitumor efficacy of the compounds. hese novel agents specifically inhibit a transcription factor that has been difficult to target with conventional agents.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C141.
Abstract C147: siRNA targeting TS and TK as an anticancer therapy
Authors: Di Cresce Christine , Figueredo Rene , Koropatnick James
Published: June 2012

Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis, repair and proliferation. Cytosolic thymidine kinase 1 (TK1) and mitochondrial thymidine kinase 2 (TK2) represent “scavenger” pathways for alternative production of essential dTMP by phosphorylation of intracellular thymidine (Thd). Both TS and TKs are upregulated in multiple human tumors, supporting a role for both in malignancy. Drugs targeting TS-protein are a mainstay in cancer treatment but toxicity to normal tissues and tumor cell drug resistance limit their effectiveness. We hypothesize that, when TS-protein is inhibited, the capacity of TKs to generate dTMP may modulate resistance to anti-TS drugs.

Antisense molecules, including oligonucleotides (ODNs) and siRNAs, can selectively target specific mRNAs (including TS and TK mRNAs) encoding proteins mediating drug resistance. We have previously reported that antisense ODNs targeting TS inhibit human tumor cell growth in vitro and in immunocompromised host mice, and antisense TS ODNs and siRNAs enhance the growth inhibitory effects of TS-targeting drugs. Antisense TS, therefore, is an attractive candidate for development as an anticancer drug, both alone and in combination with drugs targeting TS protein (raltitrexed, pemetrexed, 5FUdR, and others). The ability of antisense TS drugs to inhibit tumor cell growth and response to other TS-targeting drugs does not correlate with the degree of antisense downregulation of TS mRNA, suggesting that factors other than TS attenuate antitumor effects.

We used siRNAs targeting TK1 or TK2 to knock down TK expression in attempt to reduce human tumor cell growth when targeting TKs alone, in combination with siRNAs targeting TS, and in combination with TS siRNA and the anti-TS protein drug 5FUdR. We report that TK and TS levels are highly variable among a panel including human cervical (HeLa) and breast carcinoma (MCF7), colorectal cancer (HT-29) and human mesothelioma cell lines. Furthermore, 24 hrs post-transfection, siRNAs targeting TK1 or TK2 reduce targeted TK-mRNA by greater than 85% at concentration of 5 nM in human cervical carcinoma (HeLa) and human breast carcinoma (MCF7) cells. At 96 hrs post-transfection, siRNA targeting TK1 or TK2 do not independently reduce human tumor cell growth in vitro. Furthermore, simultaneous siRNA targeting of TS and TK effectively, independently, and non-antagonistically reduce both TS and TK mRNA. Antisense targeting TK1 or TK2 alone, unlike siRNA targeting TS alone, does not enhance the capacity of 5FUdR to inhibit human tumor cell growth in vitro. We suggest, however, that combined antisense targeting of TS and TK1/TK2 (particularly under conditions where extracellular thymidine levels are high) will be more effective than either used alone to reduce tumor cell growth and sensitize them to the effects of antitumor TS-targeting chemotherapeutics.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C147.
Abstract C150: SOX9 modulates Wnt/beta-catenin singal in cancer cells by regulating TCF4 and LRP6 expression
Authors: Wang Hong-yun , Richardson Andrea , Balk Steven P. , Yuan Xin
Published: June 2012

Introduction: Cancers frequently exploit developmental genes/pathways to sustain their uncontrolled growth or invasion. SOX9, an important developmental transcription factor is one of the signature genes of the basal-like breast cancers. SOX9 over-expression has been demonstrated in various cancers and is associated with a poor prognosis. In prostate cancer, SOX9 supports tumor proliferation, angiogenesis and local invasion in xenografts. The role of SOX9 as a biomarker and molecular mechanisms of its biological functions were further explored in this study.

Methods: Immunohistochmical analysis of SOX9 protein expression was performed on a tissue microarray of 129 breast cancer samples. Gene profiles of the breast cancer samples were examined by ANOVA analysis to identify genes that are expressed at significantly higher level among SOX9-high protein expressing samples. SOX9 regulated genes were validated by their corresponding changes in expression levels to SOX9 silencing in cancer cell lines. The effect of SOX9 silencing on cancer progression was examined in the MCF10A DCIS.com xenografts.

Results: SOX9 protein expression was highly represented in the basal-like breast cancers and was associated with poor tumor grades. Among the genes correlated with higher SOX9 expression were two major components of the canonical Wnt/ -catenin signal pathway, ie TCF4 and LRP6. These genes were up- or down-regulated in various cancer cell lines in response to SOX9 overexpression or silencing. More importantly, the Wnt3a induced -catenin activity was significantly reduced when SOX9 protein was specifically down-regulated. SOX9 down-regulation in MCF10A DCIS xenografts lead to a squamous differentiation and a block in the progression from DCIS to invasive carcinoma.

Conclusion: This study identified a novel function of SOX9 in modulating Wnt/β-catenin signaling by regulating TCF4 and LRP6 expression. SOX9 enhanced Wnt/β-catenin activity in the absence of APC or β-catenin mutations may play an important role in determining tumor subtype, maintaining tumor stem cells or supporting tumor growth or progression.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C150.
Abstract C153: Both TRAIL receptor DR5 isoforms are required for the induction of cell death in human colon carcinoma
Authors: Jani Tanvi S. , DeVecchio Jennifer , Peters Kristi , Houghton Janet
Published: June 2012

Colorectal cancer continues to be the third most common malignancy in the United States. This disease remains resistant to treatment, with only 25–30% of patients disease-free long term; for the rest, median su rvival is only 18–22 months. We previously demonstrated synergistic interaction between FUra/LV combined with IFN- (which potentiates FUra/LV-induced DNA damage and requires the death receptor Fas) in human colon carcinoma cell lines and xenografts. FUra/LV/IFN- also demonstrated responses in heavily pretreated and previously untreated patients in Phase I trial and is currently completing Phase II evaluation in Stage IV colorectal cancer. Further, lexatumumab, an agonistic TRAIL-R2 (DR5) monoclonal antibody, is highly synergistic with FUra/LV/IFN- in xenograft models, dependent on TRAIL receptor activation. Lexatumumab demonstrated the broadest spectrum in vitro activity in a panel of 6 human colon carcinoma cell lines when compared to the activity of the recombinant ligand, TRAIL (targeting DR4 and DR5).

To further understand the mechanism of lexatumumab-induced cell death via DR5, DR5 expression, was determined in human colon carcinoma cell lines (Western, RT-PCR) and primary colon tumors (RT-PCR). The short isoform of the receptor (DR5S) was expressed at a higher level than the long isoform (DR5L). To determine the specific function of each isoform in regulating death signaling, a BJAB-derived cell line lacking DR5 expression (DR5−/−) was employed. DR5 −/− cells were resistant to lexatumumab-induced apoptosis, even when each DR5 isoform was expressed individually, despite the fact that the individual isoforms could still bind ligand (lexatumumab). Analysis of receptor complex formation indicated that DR5, FADD, and caspase-8 were recruited; however, caspase-8 was not processed. Apoptotic signaling was restored when both DR5 isoforms were re-expressed in DR5−/− cells. Lexatumumab sensitivity of HCT8 cells was eliminated by stable shRNA knockdown of both DR5 isoforms, as was the recruitment of FADD and caspase-8 to the receptor complex. In contrast, following 1 hr lexatumumab treatment in wild type HCT8 cells, both DR5 isoforms were recruited to the receptor complex; cell death correlated with recruitment of FADD, cleavage of caspase-8, cleavage of RIP, and reduced expression of c-FLIPS and c-FLIPL. TRAF2 and c-IAP2, but not TRAF1 or c-IAP1, were also recruited; however, the significance of these signaling molecules in the cell death response is currently unknown.

Overall, data indicate that both DR5S and DR5L are required for lexatumumab-induced cell death via the DR5 receptor. Although the significance of this is currently unknown, factors that regulate lexatumumab-induced cell death vs survival at the level of receptor complexes are currently being elucidated.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C153.
Abstract C180: A novel bispecific platform for potent redirected killing of B-cell lymphoma
Authors: Moore Paul A. , Huang Ling , Rainey Jonah , Zhang Wenjun , Burke Steve , Yang Yinhua , Gorlatov Sergey , Li Hua , Alderson Ralph , Tuallion Nadine , Bonvini Ezio , Johnson Syd
Published: June 2012

Despite significant advances in the treatment of B-cell lymphoma, in many cases the disease remains untreatable or displays recurrence after initial response to therapy. The recent clinical success of bi-specific molecules capable of redirecting T-lymphocyte-mediated cytotoxicity (Bi-specific T-cell Engagers, BiTEs) against B-cell lymphoma provides a feasible therapeutic option. We have developed an alternate bi-specific platform of Dual-Affinity Re-Targeting (DART) molecules that is well suited for clinical applications. Compared to BiTEs, which comprise a pair of single-chain Fv domains in a single polypeptide, DARTs contain two separate chains associated into a heterodimeric, covalently linked, ‘diabody’ structure. We show here that DARTs are very stable in vitro and can be built to recruit and retarget different effector cells, including T cells or CD16- (Fc RIII)-positive cells, against B-cell-lineage targets. A CD3xCD19 DART capable of recruiting cytotoxic T cells against CD19-positive targets mediated extremely potent killing of human peripheral blood B lymphocytes or B-cell lymphoma lines in vitro, with an EC50 that compares favorably to that of a BiTE with identical Fv sequences. Redirected killing of Daudi, Raji, or Nalm6 lymphoma lines was observed in the picomolar range using either freshly purified T cells or peripheral blood mononuclear cells (PBMC) as effector populations. In contrast, no killing was observed against a CD19-negative cell line (RPMI-8226) with either resting or stimulated effector populations, indicating a requirement for CD19 engagement for T-cell-mediated killing by the CD3xCD19 DART molecule. Furthermore, a DART constructed with a humanized version of a pan anti-TCR-beta antibody (TCRxCD19 DART) was as efficient against CD19-positive targets as the CD3-based DART. A CD16xCD32B DART was also constructed to redirect CD16-positive NK and/or mononuclear phagocyte effector cells against CD32B-positive B cells, displaying potent killing against Daudi, Raji or RPMI-8226 cells but not against a CD32B-negative cell line (Ramos). Both TCR- and CD16-based DARTs show potent activity in vivo in several mouse models, including autologous B-cell depletion in huCD32B-transgenic mice, tumor reduction in human Raji Burkitt's lymphoma cell xenografts and Raji cell implants in NOD/SCID mice reconstituted with human PBMC. In conclusion, DARTs represent a robust and flexible platform to recruit different cell populations via various cell surface targets for re-directed killing applications against B-cell malignancies.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C180.
Abstract C181: Preclinical efficacy and pharmacodynamics of SCH 900105 (AV-299) an anti-HGF antibody in an intracranial glioblastoma model
Authors: Meetze Kristan A. , Connolly Kelly , Boudrow Andrea , Venkataraman Sriram , Medicherla Satya , Gyuris Jeno , Han May
Published: June 2012

Hepatocyte growth factor (HGF) is a pluripotent growth factor produced predominantly by mesenchymal or stromal cells, and binds to the well-characterized tyrosine kinase receptor, c-Met. The HGF/c-Met pathway is frequently deregulated in different types of human cancers and is thought to play an important role in regulating tumor growth, invasion, metastasis and drug resistance. HGF/c-Met autocrine and paracrine loops have been reported in a number of human cancers including breast, lung, bladder, gastric, head and neck, glioma, multiple myeloma, leukemias, and certain sarcomas.

SCH 900105, formerly known as AV-299, is a potent, humanized anti-HGF antibody. It is currently in phase 1 clinical trials that demonstrated good safety and tolerability. It has been shown to neutralize HGF binding to c-Met and inhibits its biological function in vitro, such as cell signaling, growth, motility, invasion and drug resistance. SCH 900105 was also shown to have potent anti-tumor activity in autocrine and paracrine GBM, NSCLC, pancreatic and multiple myeloma xenograft models both as monotherapy and in combinations with chemotherapeutics or targeted agents.

In vivo efficacy of systemically administered SCH 900105 was evaluated in an intracranial autocrine U87MG model. In these studies, SCH 900105 treatment resulted in significant survival benefit over an IgG treated control group. Immunohistochemistry staining for SCH 900105 in the intracranial tumor tissue suggested adequate tumor penetration of the antibody. Treatment also led to significantly decreased tumor phospho-c-Met, increased cleaved caspase-3, as well as decreased Ki67 and CD31 staining. Greater survival benefit was also seen when SCH 900105 was combined with temozolomide in U87 intracranial model. These findings suggest evaluating SCH 900105 in GBM is warranted.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C181.
Abstract C194: In vivo characterization of AMG 900, an orally active small molecule inhibitor of aurora kinases in phase 1 clinical trials
Authors: Bush Tammy L. , Ziegler Beth , Chung Grace , McElroy Patricia L. , Kendall Richard , Radinsky Robert , Geuns-Meyer Stephanie , Payton Marc
Published: June 2012

Background: The aurora family of serine-threonine kinases (Aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora kinases A and B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora kinase C function appears restricted to male meiosis. Aurora kinase B is responsible for the direct phosphorylation of histone H3 on serine-10 (p-histone H3). Aurora kinase A and B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. The emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis has focused substantial interest in developing selective small molecule inhibitors for the treatment of human cancers. AMG 900 is a novel ATP competitive small molecule inhibitor that is highly potent and selective for aurora kinases A, B, and C.

Objective: The aim of this study was to characterize the in vivo effects of AMG 900 by establishing a pharmacokinetic-pharmacodynamic relationship and profiling its antitumor activity across multiple human xenografts models using a variety of dosing schedules.

Results: In nude mice, oral administration of AMG 900 inhibited phosphorylation of histone H3 in tumors and bone marrow in a time- and dose-dependent manner. The degree of p-histone H3 suppression in cells correlated with plasma drug concentrations. The effect of AMG 900 was tested in a panel of human tumor xenograft models. Oral administration of AMG 900 at 15 mg/kg BID for two consecutive days per week or 3 mg/kg BID every day inhibited tumor growth (50–97% inhibition; p ≤ 0.0044) in all nine of the xenograft models tested compared with the vehicle control group. Importantly, three of these xenograft models are resistant to paclitaxel, docetaxel, and doxorubicin. Mice treated with efficacious doses of AMG 900 showed a transient loss of body weight (0–10%) and bone marrow cellularity, consistent with its on-mechanism effects on normal proliferating cells. Since aurora kinase inhibitors are known to cause neutropenia, studies were performed to investigate the effects of prophylactic treatment of PEGylated-G-CSF (SD/02). In mice, administration of SD/02 shortened the duration of AMG 900-induced neutropenia.

Conclusion: Based on these preclinical activities, AMG 900 has the potential to treat advanced cancers, including tumors resistant to standard-of-care chemotherapeutic agents such as paclitaxel and docetaxel. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C194.
Abstract C196: Co-treatment with NVP-BEZ235 and heat shock protein (hsp) 90 or pan-deacetylase (DAC) inhibitor is synergistically active against non-small cell lung cancer (NSCLC) cells with mutant RAS and LKB1
Authors: Upadhyay Sunil , Fiskus Warren C. , Rao Rekha , Joshi Atul , Koul Sanjay , Balusu Ramesh , Mudunuru Uma , Buckley Kathleen M. , Quadt Cornelia , Atadja Peter , Garcia-Echeverria Carlos , Bhalla Kapil N.
Published: June 2012

Inactivating somatic mutation of LKB1 is found in approximately 30% of NSCLC and is often coincident with activating K-RAS mutation. In a mutant K-RAS-driven model of mouse lung cancer, strong cooperation and aggressive phenotype was seen with hemizygous inactivation of LKB1. Loss of LKB1 results in activation of mTOR pathway while somatic activating mutation in K-RAS results in activation of PI3K/AKT and RAS/RAF/ERK. Therefore therapeutic stratagies which target these pathways may have significant affect on the outcome of these cancers. NVP-BEZ235 is a dual PI3K and mTOR inhibitor and panobinostat (PS) (Novartis Pharmaceuticals Inc) is a pan-histone deacetylase (HDAC) inhibitor, which has been shown to induce proteasomal degradation and depletion of the levels of hsp90 client proteins, including AKT and c-RAF. Here, we determined the effects of BEZ235 and/or PS or AUY922 (an hsp90 inhibitor) on NSCLC A549 and H460 cells with LKB1 and K-RAS mutations (A549 and H460). Treatment with BEZ235 (200 to 1000 nM), PS (25 to 50 nM) or AUY922 (25 to 100 nM) dose-dependently increased % of cells in G2/M and decreased % of cells in S phase of cell cycle, as well as induced apoptosis of the NSCLC cells. Co-treatment with AUY922 or PS increased BEZ235 mediated depletion of p-AKT, p-4EBP1 and p-p70S6K levels, as well as synergistically induced apoptosis of A549 and H460 cells (combination indices < 1.0). Combined therapy with BEZ235 (25 mg/Kg) and PS (10 mg/Kg) or AUY922 (20 mg/Kg) resulted in significantly more tumor growth delay than treatment with each of the agents alone of A549 cell xenografts in nude mice (p < 05). We also determined the activity of BEZ235 and/or PS in the NSCLC H368 cells with mutant K-RAS and either wild-type (H358 cells) or stable knock down of LKB1 expression (H358/LKB1-KD cells), achieved by shRNA against LKB1, mimicking the presence of mutant LKB1. As compared to each agent alone, co-treatment with BEZ235 and PS exhibited a higher level of antitumor synergy against H358/LKB1-KD versus H358 cells. These pre-clinical in vitro and in vivo studies demonstrate that combined treatment with the dual PI3K/mTOR inhibitor BEZ235 and PS or AUY922 could potentially be a promising targeted therapy for NSCLC that harbors K-ras and LKB1 mutations.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C196.
Abstract C202: Targeting the mitotic checkpoint for cancer therapy with NMS-P715, a novel MPS1 kinase inhibitor
Authors: Colombo Riccardo , Caldarelli Marina , Mennecozzi Milena , Giorgini Maria L. , Sola Francesco , Cappella Paolo , Ballinari Dario , Perrera Claudia , Re Depaolini Stefania , Rusconi Luisa , Avanzi Nilla , Volpi Daniele , Bossi Roberto , Bertrand Jay A. , Pesenti Enrico , Isacchi Antonella , Donati Daniele , Moll Jurgen
Published: June 2012

The Spindle Assembly Checkpoint (SAC) is a mitotic mechanism specifically required for proper chromosomal segregation ensuring that cells do not divide until all sister chromatids correctly align to the metaphase plate. MPS1 kinase, (also known as TTK) is a key regulator of SAC functions and it has been found to be up-regulated in a number of tumors of different origins. The hypothesis is that SAC activity could be highly required to sustain aneuploid tumor growth and MPS1 inhibitors may have a therapeutic benefit in the treatment of certain cancers.

We have identified NMS-P715, a potent and selective oral bioavailable small-molecule MPS1 kinase inhibitor with an ATP-competitive bindingmode. Treatment of cells with NMS-P715 accelerates mitotic exit with an IC50 of 53 nM. This is accompanied by reduction of the mitotic length, MPS1 dephosphorylation, chromosomal mis-alignment, delocalization of kinetochore components and massive aneuploidization, which ultimately leads to cell death.

Proliferation data performed on a large panel of 126 cell lines shows a wide range of activity and indicates selective activity against tumoral cells compared to normal cells. Oral administration of NMS-P715 in tumor xenografted mice resulted in potent tumor growth inhibition in an ovarian cancer xenograft model and was accompanied by biomarker modulation, confirming the expected MOA.

Our data provide evidence that inhibition of MPS1 kinase and SAC abrogation could represent a new promising approach to cancer therapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C202.
Abstract C209: Inhibition of both c-Met and EGFR signaling shows synergistic antitumor activity in non-small cell lung cancer model
Authors: Tammam Jennifer G. , Davis Lenora , Ware Chris , Reilly John F. , Deshmukh Sujal V. , Dinsmore Christopher , Marshall Gary , Harrington Elizabeth A. , Pan Bo-Sheng , Lutterbach Bart A. , Majumder Pradip K.
Published: June 2012

c-Met and ErbB family of receptors including EGFR, ErbB-2, ErbB-3 and ErbB-4 are co-expressed in many human tumors including Non-Small Cell Lung Cancers (NSCLC). Several reports suggest that the activation of c-Met and ErbB family receptors is associated with poor prognosis in multiple tumors types. Recently it has been shown that activation of c-Met is common in Erlotinib and Gefitinib-resistant NSCLC patients. Combining c-Met and EGFR inhibitors has been proposed as a strategy for treatment in EGFR inhibitor-refractory lung cancers. To understand the cooperation of these pathways, we used a c-Met activated human NSCLC cell line (EBC-1), where EGFR signaling network is also active. In vitro treatment of EBC-1 cells with a combination of Erlotinib and MK-8033 (an inhibitor of c-Met and RON kinases) resulted in greater growth inhibition relative to treatment with either agent alone. In order to validate these in vitro data, we have tested MK-8033 and EGFR inhibitors (Erlotinib and Gefitinib) in vivo. The data suggests the effect of MK-8033 with Gefitinib or Erlotinib on tumor growth is greater when combined as compared to monotherapy in this preclinical xenograft model. PK data suggests no drug-drug interaction of MK-8033 and EGFR inhibitor when administered together. Exposure of these agents was sufficient to inhibit both kinases in tumors. Our studies provide a rationale for the combination of MK-8033 and Erlotinib or Gefitinib in EGFRi refractory patients with NSCLC.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C209.
Abstract C219: Molecular and cellular pharmacology of the novel non-camptothecin topoisomerase I inhibitor, GENZ644282
Authors: Sooryakumar Dhriti , Dexheimer Thomas , Teicher Beverly A. , Pommier Yves
Published: June 2012

Camptothecin (CPT) derivatives are powerful anticancer drugs because of their ability to trap topoisomerase I (Top1)-DNA cleavage complexes. However, they exhibit clinical limitations due to the instability of their α-hydroxylactone six-membered E-ring structure, which is essential for trapping Top1-DNA cleavage complexes. Additionally, they exhibit bone marrow and intestinal toxicity in adults and are drug efflux substrates. Here, we report a Top1 inhibitor, Genz644282. In the cell lines studied, we find that Genz644282 as well as its metabolites induce Top1 cleavage at similar, as well as unique genomic positions, compared with CPT. The compound also induces protein-linked DNA breaks and Top1-DNA cleavage complexes that persist longer after drug removal than CPT. Concentration-dependent and persistent H2AX formation was readily observed in cells treated with Genz644282, and was present in greater than 50% of the cell population following 24 hours drug treatment. The compound shows partial cross-resistance in cell lines resistant to CPT. These cell lines include the prostate cancer cell line (DU145RC0.1), and human leukemic (CEM/C2) cells. Genz644282 demonstrated limited cross-resistance in Top1-knockdown colon cancer (HCT116) and breast cancer (MCF7) cells. The compound also demonstrated limited resistance in human lung cancer cells (H460/MX20). These lung cancer cells express (BCRP, ABCG2), a member of the ATP-binding cassette family of cell surface transport proteins known to have ABCG2 drug-efflux pumps.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C219.