bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B5: ACE-041, a soluble activin receptor-like kinase-1 fusion protein, is a novel antiangiogenic agent
Authors: Mitchell Dianne , Pobre Eileen G. , Mulivor Aaron W. , Grinberg Asya V. , Castonguay Roselyne , Monnell Travis E. , Solban Nicolas , Ucran Jeff , Pearsall R. Scott , Underwood Kathryn W. , Seehra Jasbir , Kumar Ravindra
Published: June 2012

The therapeutic targeting of angiogenesis has been associated with clinical benefit for a number of tumors including colorectal carcinoma, non-small cell lung cancer, breast cancer, renal cell carcinoma and glioblastoma. Activin receptor-like kinase-1, an endothelium specific member of the TGFβ-type I receptor family, is believed to play a role in modulating angiogenesis. Homozygous inactivation of Alk1 in mice leads to embryonic lethality resulting from severe vascular abnormalities, while heterozygous mice exhibit chronic hemorrhaging and arteriovenous malformation similar to hereditary hemorrhagic telangiectasia-2 (HHT-2) patients harboring mutations in ALK1. To examine the potential of ALK1 inhibition as a novel anti-angiogenic therapy for cancer, we developed a soluble form of the ALK1 receptor (ACE-041) by fusing the extracellular region of human ALK1 to the Fc of human IgG1. ACE-041 was shown to bind only BMP-9 and BMP-10 out of 29 ligands tested in TGFβ family. ALK1 inhibition blocks SMAD 1/5/8 signaling and blocks in vitro cord formation of endothelial cells. ACE-041 inhibited in vivo blood vessel formation using Matrigel plug assay and chick chorioallantoic assay (CAM). Moreover, ACE-041 decreased tumor volume in a modified tumor CAM assay and in an orthotopic breast cancer model induced by MDA-MD-231 and MCF-7 mammary adenocarcinoma cells. These data suggest that ALK1 inhibition may be a promising therapeutic strategy for patients with breast and other solid tumors and support advancement of ACE-041 into clinical trials.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B5.
Abstract B51: A phase I dose-escalation study of MGCD265 in patients with advanced malignancies (study 265-102)
Authors: LoRusso Patricia , Kurzrock Razelle , Weise Amy , Heath Elisabeth , Drouin Michel , Juretic Manuela , Maroun Christiane , Karam Andre , Mehran Mariam , Besterman Jeffrey , Martell Robert , Wheler Jennifer
Published: June 2012

Introduction: MethylGene has developed a small molecule inhibitor, MGCD265, that inhibits c-Met, Ron, all three members of VEGFR family and Tie-2, thereby inhibiting multiple pathways involved in tumor growth, metastasis and angiogenesis. The primary objectives of this study are to determine the maximum tolerated dose and the safety profile of MGCD265 administered with interruption to patients with advanced malignancies.

Methods: This is a multicenter, open-label, dose escalation study of oral MGCD265 administered once daily on a 1 week on/1 week off schedule of each 28-day cycle. Cohorts of 3–4 patients were enrolled per dose level, initially with dose doubling between cohorts, followed by smaller increments once grade 2 drug-related toxicity was observed. Dose limiting toxicity (DLT) was defined as: grade 4 neutropenia, grade 4 thrombocytopenia, any ≥ grade 3 nonhematologic toxicity, severe/sustained hypertension or any toxic effect leading to a patient missing ≥ 4 doses of MGCD265. Treatment continued until disease progression or toxicity. Response, as a secondary objective, was assessed every 2 cycles by RECIST criteria.

Results: Twenty patients (20) have been recruited to date: 4 patients in cohort 24 mg/m2; 4 patients in cohort 48 mg/m2; 3 patients in cohort 96 mg/m2; 3 patients in cohort 192 mg/m2; 3 patients in cohort 255 mg/m2; and 3 patients in cohort 340 mg/m2. The overall age range is 40 to 72 years and the gender distribution among the cohorts is 13 female to 7 male patients. No DLTs have been observed to date but grade 2 adverse events (AE) considered at least possibly related to the treatment included diarrhea (n=2), nausea (n=1) and intermittent dizziness (n=1). No grade 3 or higher drug-related AE have been reported. To date, 5 patients have experienced stable disease per RECIST. One patient with a sarcomatoid bladder cancer (dosed at 96 mg/m2), who was progressing prior to study entry, has been treated for 12 cycles. An archived biopsy obtained prior to MGCD265 treatment from this patient shows c-Met expression and autophosphorylation. One patient with medullary thyroid cancer experienced tumor shrinkage (−10%).

Conclusions: To date, intermittent administration of MGCD265 was found to be well tolerated with preliminary signs of activity. Recruitment of additional cohorts is underway.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B51.
Abstract B52: Phase 1 study of XMT-1001, a novel water soluble camptothecin conjugate, given as an intravenous infusion once every three weeks to patients with advanced solid tumors
Authors: Sausville Edward A. , Garbo Lawrence , Weiss Glen J. , Shkolny Dana , Yurkovetskiy Alexander V. , Bethune Claudette , Ramanathan Ramesh K. , Fram Robert J.
Published: June 2012

Introduction: XMT-1001 is a water soluble macromolecular conjugate of camptothecin (CPT). In this novel CPT pro-drug, CPT is conjugated with a 70 kDa biodegradable hydrophilic polyacetal, poly (1-hydroxymethylethylene hydroxymethylformal). XMT-1001 has an improved therapeutic window compared with CPT and irinotecan in human tumor xenograft models, providing a compelling rationale for its clinical development.

Methods: This is an open label, dose escalation study of XMT-1001 given as an IV infusion once every 3 weeks. The objectives of the Phase 1 study are to determine the maximum tolerated dose (MTD) and to assess safety and pharmacokinetics (PK) of XMT-1001. Initially 3 patients (pts) are entered at each dose level, with expansion to 6 pts if there is a dose limiting toxicity. Analyses of plasma and urine were performed for XMT-1001 (conjugated CPT), 2 major drug release products, and for unconjugated (free) CPT.

Results: Forty six patients with refractory solid tumors have received 141 cycles of XMT-1001 at 13 dose levels ranging from 1.0–64.2 mg CPT equivalents (equiv.)/m2. Two pts had Grade (Gr) 3 infusion reactions consistent with hypersensitivity to study drug. Symptoms reversed upon discontinuation of study drug. Upon introduction of new clinical trial material with an improved formulation, no infusion reactions suggestive of hypersensitivity have occurred (25 pts, 79 cycles). No hemorraghic cystitis or ≥ Gr 3 diarrhea was noted. Gr 2 neutropenia occurred in 1 pt treated at 15.4 mg CPT equiv./m2. Stable disease was observed in 12 of 43 evaluable pts with the following tumor types and dose levels expressed in mg CPT equiv./m2: NSCLC (1.0 mg/m2, 6 wks; 20.5 mg/m2, 30 wks to date; 36.3 mg/m2, 18 wks to date), nasopharyngeal (36.3 mg/m2, 12 wks to date), ovarian (4.9 mg/m2, 12 wks), pancreas (4.9 mg/m2, 36 wks), appendiceal (7.3 mg/m2, 12 wks), bile duct (9.1 mg/m2, 18 wks), basal cell (11.6mg/m2, 6 wks), colon (15.4 mg/m2, 18 wks), gastric (20.5 mg/m2, 12 wks), and rectal (27.3mg/m2, 6 wks). The PK of XMT-1001 (conjugated CPT) demonstrates dose proportional increases in exposure XMT-1001 and confirms the formation of its expected release products. Levels of free CPT in urine have been low.

Conclusions: 1. XMT-1001 can be given safely to patients; 2. XMT-1001 has a favorable PK profile; 3. Prolonged stable disease lasting ≥ 12 weeks was seen in 9 patients with advanced, refractory tumors at doses below MTD.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B52.
Abstract B6: A phase II study of the efficacy and safety of foretinib, a novel receptor tyrosine kinase inhibitor, given on an intermittent 5-days-on/9-days-off (5/9) schedule in patients with recurrent or metastatic squamous cell cancer of the head and neck
Authors: Seiwert Tanguy , Swann Suzanne , Kurz Hazel , Bonate Peter , McCallum Stewart , Sarantopoulos John
Published: June 2012

Foretinib (also known as GSK1363089 and XL880) is a small-molecule, spectrum-specific receptor tyrosine kinase inhibitor, with potent activity against MET, VEGFR2, and multiple additional tyrosine kinases with tumor growth-promoting and angiogenic properties. Antitumor activity has been observed in 2 previous phase I studies. This phase II study was conducted to evaluate responses in squamous cell cancer of the head and neck (SCCHN).

An open-label, single-arm, multicenter trial employing a Simon 2-stage design was conducted, with a total of 41 patients to be enrolled in the study. One or more responses in the first 14 subjects were required to progress to the second stage. Foretinib was administered as a 240-mg oral dose once daily on days 1–5 of a 14-day cycle to subjects with recurrent and/or metastatic SCCHN. Other eligibility requirements included measurable disease, Eastern Cooperative Oncology Group (ECOG) performance status (PS) = 1 or 0, and no history of uncontrolled tumor bleeding. Exclusions included more than 1 prior therapy for recurrent or metastatic disease, history of brain metastasis, and rapid progression within 6 months after curative-intent therapy for local/loco-regional disease. Endpoints were objective response rate, safety, duration of response, progression-free survival (PFS), overall survival (OS), and characterization of the pharmacokinetics. Tumor volume was assessed every 8 weeks by Response Evaluation Criteria in Solid Tumors.

This study did not meet the predefined criteria for continuing to the second stage. Response and safety data are available for 14 patients with a data cut off date of July 24 2009 (median age = 59.0; males/female = 13/1: ECOG PS 0/1 = 9/5 patients). A maximum of 30 cycles was administered (median = 4.5). The most common reason for withdrawal was progressive disease. Treatment-emergent adverse events occurring in >25% of individuals included (# [%]): fatigue (7 [50%]), constipation (5 [36%]), hypertension (5 [36%]), anorexia (4 [29%]), dysphagia (4 [29%]), weight loss (4 [29%]), increased alanine transaminase (4 [29%]), increased aspartate transaminase (4 [29%]), dyspnea (4 [29%]), headache (4 [29%]), and mucosal inflammation (4 [29%]). Investigator-assessed median PFS (95% confidence interval) was 3.65 (3.42, 5.32) months; median OS was 5.59 (3.71, not reported) months. The best overall response was stable disease (SD), with a median duration of 4.11 (3.65–13.86) months.

Foretinib 240 mg on a 5/9 schedule was generally well tolerated. SD was the best observed response. The safety and efficacy results support further investigation in combination with other targeted agents or cytotoxic chemotherapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B6.
Abstract B64: Gene expression profile of a liposarcoma mixoid cell line selected in vitro for resistance to Trabectedin
Authors: Marchini Sergio M. , Bernasconi Sergio , Erba Eugenio , Uboldi Sarah , Clivio Luca , Chiorino Giovanna , Santarsier Annapaola , Fuso Ilara , Marangon Elena , Sala Federica , Galmarini Carlos , Tercero Juan Carlos , Mantovani Roberto , D'Incalci Maurizio
Published: June 2012

Introduction: Trabectedin is a marine alkaloid originally isolated from Ecteinascidia turbinata approved in Europe for the second line of therapy in soft tissue sarcomas (STS). Here we characterize and compare the gene expression profile of 402-91/ET Res liposarcoma cells obtained after continuous exposure to trabectedin in vitro with that of their parental cell line 402-91.

Methods: Drug sensitivity was measured by colony assay and Flow Cytometric analysis was used to analyze cell cycle perturbations. Microarray analysis between sensitive and 402-91/ET Res cells was performed using commercially available arrays onto which 44K oligos were pre-spotted. Gene expression analysis was performed with “R” package software. Pathway analysis was performed using Metacore software. qRT-PCR was used for data validation. Statistical analysis was performed using Graphpad software.

Results: 402-91/ET Res cells were selected in vitro for their stable resistance to trabectedin by step-wise increase in the drug concentration (25 nM) lasting 14 months. Resistance to Trabectedin was stable over 22 months without compound exposure. Cells were cross resistant to melphalan, but more sensitive to taxanes, vinblastine, temozolomide and UV rays. Resistance was not associated to the expression of MDR proteins as well as different uptake/efflux. Cell cycle analysis revealed that trabectedin is able to induce a G2-M block in the sensitive cell line but not in the resistant one. The expression levels of the FUS-CHOP gene were comparable between the 402-91 and 402-91/ET Res cells but with a different trans-activation ability. Microarray analysis identified 511 probes set able to discriminate between sensitive and resistant cell lines. Pathway analysis revealed that most of the genes altered in the 402-91/ET Res cells were associated with liposarcoma phenotype and functionally involved in the CREB pathway as well as control of apoptosis.

Conclusion: Data suggests that in the 402-91/ET Res cells FUS-CHOP trans-activation activity has been impaired. Studies are in progress to address whether these mechanisms are implicated in sensitivity of liposarcoma cell lines to trabectedin.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B64.
Abstract B65: Overexpression of MRP4 or MRP5 confers resistance to the nucleoside analogs cytarabine and troxacitabine, but not gemcitabine
Authors: Adema Auke D. , Floor Karijn , Smid Kees , Honeywell Richard J. , Peters Godefridus J.
Published: June 2012

Multidrug resistance proteins (MRP) confer resistance to drugs from different classes. MRP4 and MRP5 are related MRP proteins and can transport organic anions. Hence their overexpression has been associated with resistance to several purine analogs, such as 6-mercaptopurine and adefovir (PMEA; 9′-(2′-phosphonylmethoxyethyl)adenine)), and antifolates, such as methotrexate (MTX). 6MP itself is not a substrate for MRP4 and 5, but its monophosphate is, in contrast to the di- (DP) and triphosphates (TP). Similarly MTX (a monoglutamate) is a substrate for various MRPs but the higher glutamates are usually not. Resistance to MTX was not observed at a long exposure because of accumulation of polyglutamates, but a 4-hr exposure to MTX conferred resistance. The aim of this study was to investigate whether MRP4 and 5 overexpression would confer resistance to the widely used nucleoside analogs cytarabine (ara-C) and gemcitabine (GEM), and the novel L-nucleoside analog Troxacitabine (TROX). As model systems we used HEK293 cells transfected with either MRP4 (HEK/MRP4; 59-fold increased MRP4 level) or MRP5 (HEK/MRP5i; 991-fold increased MRP5 level) and determined sensitivity after an exposure for 4 hr (followed by 68 hr drug-free medium) and 72 hr. Uptake and accumulation of GEM and TROX were measured with HPLC coupled to either UV or MS-MS, that of ara-C with the radioactive drug. HEK293 cells were moderately sensitive to ara-C (IC50 1.8 µM) and TROX (2.1 µM) and more sensitive to GEM (46 nM) at 72 hr exposure. At 4 hr exposure, the IC50s increased 5-, 5- and 10-fold, respectively. At 4 and 72 hr exposure a 2-3-fold resistance to TROX and ara-C was found in HEK/MRP4 cells, and a 2.5-fold resistance to ara-C in HEK/MRP5 cells. No resistance to GEM was found. PMEA showed a 3-fold resistance in both cell lines. Resistance in the MRP4/5 cells could be reversed by the specific inhibitors probenecid and indomethacine. In order to explain this resistance, accumulation and retention of the active nucleotides were evaluated. GEM-TP even tended to be higher in the MRP4/5 cells. However, accumulation of ara-C nucleotides after 4 hr was 3- and 2-fold lower in MRP4 and MRP5 cells, respectively. After washing with drug-free medium (DFM), the concentration of ara-CTP declined more rapidly in MRP4 compared to the wild-type. Although TROX accumulation was similar in the 3 cell lines, in MRP4 and MRP5 cells it decreased 4- and 2-fold faster, respectively, after the DFM period. TROX-phosphates were about 25% lower in MRP4/5 cells and decreased rapidly in MRP4, but not in MRP5 cells. In conclusion, MRP4 and MRP5 overexpression confer resistance to TROX and ara-C, but not to GEM, which was associated with a rapid decline of the ara-C and TROX-nucleotides in HEK/MRP4-5 cells.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B65.
Abstract B66: Resistance to bortezomib in myeloma cell lines is mediated by increased IGF-1/Akt signaling to activate Cot kinase and NF-κB
Authors: Kuhn Deborah J. , Bjorklund Chad C. , Orlowski Robert Z.
Published: June 2012

Background: Targeting the proteasome as an anti-cancer therapy was first realized with bortezomib, now one of the standards used in relapsed/refractory multiple myeloma and mantle cell lymphoma. Despite this advance, response rates have been as low as 23%, indicating the importance of innate or acquired resistance to bortezomib.

Methods: To improve our understanding of the mechanisms responsible for bortezomib resistance, we developed cell line models of interleukin (IL)-6-dependent (ANBL-6 and KAS-6/1) and -independent (RPMI 8226 and OPM-2) bortezomib-resistant (BR) myeloma by continuously exposing drug-naïve cells to increasing bortezomib concentrations. All of the lines where characterized by gene expression profiling to identify potential mechanisms of resistance.

Results: Cell lines resistant to bortezomib retained their viability in the presence of 50 nM or more of bortezomib with degrees of resistance up to 4.3. Gene expression profiling revealed up-regulation of insulin-like growth factor (IGF)-1 transcripts (5.2-fold), Akt (3.2-fold), and cancer Osaka thyroid (Cot) kinase (2.3-fold). Consistent with our profiling data, BR clones had increased soluble IGF-1 levels, increased IGF-1 protein, and phospho-IGF-1R activation. Next we examined the effect of overexpression of Cot on BR clones. Cot functions to regulate nuclear factor kappa B (NF-κB)-dependent transcription in response to Akt by promoting post-translational processing of inactive NF-κB p105 to active p50. Basel levels of nuclear activated p50 were increased up to 4.7-fold in BR clones. Activated NF-κB dimers were also found to be significantly increased in ANBL-6.BR and 8226.BR cells as evidenced by phosphorylation of p65 (RelB). Our examination into Cot expression in drug-naive and BR cells showed a large increase of Cot protein and phospho-Cot (Thr290) activation in 8226.BR cells that was enhanced with 24-hour bortezomib treatment. A transcriptional target of NF-κB is Bcl-2 was also found to be increased in profiling studies (5.5-fold) and correlates with increased expression in BR cell lines tested. Treatment with 10 nM bortezomib for 6 hours led to a decreased in Bcl-2 protein both drug-naïve and drug-resistant cells. A decrease in Bcl-2 protein was also observed in ANBL-6.BR, RPMI 8226.BR, and OPM-2.BR cells in response to 24-hour treatment with picropodophyllin, a pharmacologic inhibitor of IGF-1R activation, consistent with the hypothesis that the IGF-1/Akt signaling axis in important in Bcl-2 protein expression in bortezomib-resistant clones.

Conclusions: Our data support the hypothesis that increased signaling through the IGF-1/Akt axis and enhanced expression of Cot kinase is another mechanism that mediates stable bortezomib resistance in vitro through the activation of NF-κB and its downstream transcriptional targets, including Bcl-2.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B66.
Abstract B73: Lapatinib-mediated radiosensitization of EGFR+/basal breast cancer correlates withERK inhibition in vivo and requires MEK/ERK inhibition in vitro
Authors: Sambade Maria J. , Kimple Randall J. , Camp Terese , Peters Eldon , Livasy Chad A. , Sartor Carolyn I. , Shields Janiel M.
Published: June 2012

To determine whether the dual EGFR/HER2 kinase inhibitor lapatinib can radiosensitize EGFR+ breast cancer in vivo, mice bearing xenografts of basal-like/EGFR+ SUM149 breast cancer cells were treated with lapatinib and fractionated radiation therapy over a 10 d period and tumor growth inhibition determined and correlated with changes in activation of downstream signaling to ERK1/2 and AKT by immunohistochemistry. Basal-like/EGFR+ SUM149 breast cancer tumors were completely resistant to treatment with lapatinib alone but highly growth impaired with lapatinib + radiation exhibiting an enhancement ratio average of 2.75 and a fractional tumor product ratio average of 2.06 over the duration of the study. Immunohistochemical analysis demonstrated that radiosensitization by lapatinib correlated with inhibition of ERK1/2 but not AKT. In vitro analysis of other basal breast cancers consistently demonstrated a loss of radiation induced MEK1/2 inhibition with lapatinib treatment. Treatment of the SUM102 basal-like/EGFR+ cell line with the MEK inhibitor CI1040 resulted in radiosensitization at levels similar to that seen with lapatinib alone. Furthermore, constitutive activation of RAF in SUM102 cells abolished the ability of lapatinib to radiosensitize these cells and resulted in enhanced radioresistance relative to parental cells. Collectively, this work demonstrates that lapatinib radiosensitizes EGFR+/basal-like breast cancer both in vitro and in vivo in a MEK/ERK dependent manner, and suggests that lapatinib in combination with fractionated radiation therapy may provide an effective treatment option for breast cancers of this subtype.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B73.
Abstract B77: Erk phosphorylation predicts synergism between gemcitabine and EGFR inhibitor AG1478
Authors: Peter Luk P. , Links Matthew J. , Galettis Peter
Published: June 2012

Clinical trials combining epidermal growth factor receptor (EGFR) inhibitors with gemcitabine-based chemotherapy in non-small cell lung cancer (NSCLC) have not produced a survival advantage. This may be caused by antagonism between the two drugs or mutations that promote such, possibly KRAS mutation. It has been hypothesised that this antagonism relates to G1 arrest caused by EGFR inhibition. This study aimed to explore the relationship between synergy/antagonism, G1 arrest and phospho-ERK, as a measure of mitogen-activated protein kinase (MAPK) inhibition. Two NSCLC cell lines, A549 (mutant KRAS) and H322 (wildtype KRAS), were treated with gemcitabine and the EGFR inhibitor AG1478 alone and concurrently. Cytotoxicity was assessed using SRB assay and cytotoxic interaction was calculated using median effect analysis. Cell cycle distribution was assessed using flow cytometry and ERK phosphorylation was determined by ELISA. Cytotoxic interaction between gemcitabine and AG1478 were dose dependent with antagonism at high dose AG1478, particularly in the KRAS mutant cell line. Under conditions in which gemcitabine caused S phase arrest, antagonism was related to the G1 arrest effects of AG1478. However, synergy was also observed in the presence of gemcitabine-induced g1 arrest. Furthermore, gemcitabine increased ERK phosphorylation, the suppression of which by AG1478 was related to antagonism particularly in KRAS wildtype cell line. In the KRAS mutant cell line, synergy/antagonism was observed without a changed in phosphorylation of ERK. Our findings are consistent with a model in which gemcitabine causes cell cycle arrest and increased ERK phosphorylation. Low dose AG1478 is synergistic possibly due to suppression of EGFR-related survival signals. However, anti-proliferative effect of high dose AG1478 is antagonistic as it interferes with gemcitabine-induced cell cycle arrest as well as reduces ERK phosphorylation, which may be an important death signal. This data confirms the importance of EGFR inhibition-related G1 arrest in antatogism but also identifies that synergy can still occur in the presence of G1 arrest and highlights an important potential role for phospho-ERK in determining cell death. In summary, this study suggests that ERK phosphorylation is more predictive of synergism/antagonism than cell cycle changes when gemcitabine is combined with an EGFR inhibitor.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B77.
Abstract B81: AUY922, a heat shock protein 90 inhibitor, actively inhibits human gastric cancer cells via down regulation of growth factor receptors, and acts synergistically with relevant chemotherapeutic agents
Authors: Yeh Kun-Huei , Gao Chiu-Hua , Lin Yu-Lin , Cheng Ann-Lii
Published: June 2012

Heat shock protein 90 (Hsp90) is the most abundant cellular chaperone and has nearly 100 pivotal client proteins, which are commonly dysregulated in cancers. The positive rate of Hsp90beta in gastric cancer tissue was approximately 30%, and was higher than non-cancerous gastric mucosa. Hsp90 inhibitors should have great potentials in the exploration for treatment strategy in gastric cancer (GC).

AUY922, kindly provided by Novartis Pharma AG, is a novel resorcinylic isoxazole amide Hsp90 inhibitor. A panel of human GC cell lines (N87, AGS, SNU-1, SNU-16, KATO-III from ATCC) was used in this study. Using 72-hour MTT colorimetric assay, we demonstrated that AUY922 has highly active growth inhibitory effects as a single agent of IC50 in the ranges between 8.1 ± 0.2 and 14.7 ± 0.8 nM (except the c-met-overexpressing KATO-III cells with a IC50 of 158.1 ± 5.1 nM), which is more active than 17-AAG (17-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethyl-amino-17-demethoxygeldanamycin) of IC50 between 17.2 ± 0.6 and 61.3 ± 1.9 nM, and IC50 between 10.5 ± 0.5 and 24.3 ± 1.0 nM, respectively. By MTT colorimetric assay and median effect analysis, AUY922 has significant synergistic cytotoxicity with cisplatin, oxaliplatin, 5-fluorouracil (5-FU), and irinotecan, while acts antagonistically with paclitaxel and docetaxel in GC cells.

Several important growth factor receptors are highly expressed in GC, namely 50-60% of EGFR (epidermal growth factor receptor, HER1), 15–22% of HER-2/neu, and 32–58% of c-met (hepatocyte growth factor receptor, HGF receptor), respectively. By immunoblotting analysis, AUY922 effectively down-regulates growth factor receptors (c-met, EGFR, and HER-2/neu) with a concentration of 25nM, 25nM, and 125nM in KATO-III GC cells, respectively. The c-met-overexpressing KATO-III cells are highly resistant to AUY922. In KATO-III cells, knocking-down of c-met by siRNA, then examining by 48-hour MTT colorimetric assay may partly abrogate the AUY922 resistance and increase sensitivity to AUY922 by decreasing IC50 from 790.0 ± 10.6 nM to 350.8 ± 5.4 nM.

Our data indicate that AUY922 actively inhibits human GC cells via down regulation of growth factor receptors, and acts synergistically with various relevant chemotherapeutic agents. It is reasonable to use AUY922 alone, or to combine AUY922 with relevant chemotherapeutic agents (cisplatin, oxaliplatin, 5-FU, or irinotecan) in future development of clinical trials for gastric cancers.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B81.
Abstract B85: Structure and function of a novel Bag1-L derived peptide that inhibits prostate tumor cell growth
Authors: Neeb Antje , Maddalo Danilo , Muhle Claudia , Jehle Katja , Wenzel Wolfgang , Cato Andrew CB
Published: June 2012

Peptide ligands that specifically target tumor cells represent useful tools for cancer therapy. They can be used for the transport and specific delivery of cell death-inducing sequences or chemotoxic drugs to tumor cells. Here we describe a peptide from a conserved region of Bag-1 (Bcl-2 associated athanogene-1) proteins that induces cell death without the need for a pro-drug conjugate or fusion with a death-inducing sequence. Stable expression of the Bag-1 peptide in 22Rv.1 prostate tumor cells interferes with tumor cell growth and survival in vivo and in vitro and elevates the level of apoptosis in tumor cells treated with the topoisomerase inhibitor II etoposide. Structural analysis using NMR and circular dichronism (CD) show that this peptide is completely unstructured. Nevertheless it interacts with specific sequences of the glucose regulated proteins GRP75 and GRP78. Domain mapping studies identified the ATPase domain of GRP78 and the substrate-binding domain of GRP75 as interaction sites of the Bag1-peptide. Both GRP75 and GRP78 belong to the heat shock protein family and act as protective chaperones in the unfolded protein response. In addition both proteins are over expressed in many human tumors and are found exclusively on the surface of tumor cells. In particular, the GRP78 protein that is present on the surface of tumor cells is glycosylated. Using deglycosylation and GST-pull down analysis, we can show that the Bag1-peptide also binds to the glycosylated form of GRP78 indicating its ability to target tumor cells by binding to the cell surface. Thus we have identified a candidate peptide for a broad-based tumor therapy that function by interfering with the action of GRP75 and GRP78.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B85.
Abstract B90: Understanding the effects of RAF inhibitors on RAF signaling in B-RAF V600E mutant versus wild type tumors
Authors: Yen Ivana , Song Kyung , Stokoe David , Brandhuber Barbara , Gloor Susan , Aliagas Ignacio , Anderson Daniel , Morales Tony , Vigers Guy , Ludlam Mary , Alvarado Ryan , Liu Bonnie , Sideris Steve , Ramaswamy Sreema , Heise Christopher , Belvin Marcia , Friedman Lori , Hatzivassiliou Georgia , Malek Shiva
Published: June 2012

Constitutive activation of the Ras, RAF, mitogen-activated protein kinase (MEK), and extracellular signal regulated kinase (ERK) pathway is a common finding in many human cancers. Activating mutations in K-Ras or B-RAF constitute over 30% of all mutations in human tumors. Therefore, identifying therapeutic strategies to downregulate this pathway could have broad therapeutic impact. It has been demonstrated that RAF inhibitors selectively inhibit B-RAFV600E tumors and not Ras mutant tumors, despite functioning as one of the key effector enzymes downstream of Ras and upstream of MEK. In this work we demonstrate that although RAF inhibitors effectively inhibit phospho-MEK and phospho-ERK levels in B-RAFV600E cells, pathway induction is observed for RAF inhibitors in B-RAF WT/K-Ras Mutant or B-RAF WT/K-Ras WT cell lines as evidenced by increases in phospho-MEK, phospho-ERK and phospho-p90RSK levels. We also show that with increasing concentrations of RAF inhibitors, the downstream pathway can be inhibited in WT B-RAF and mutant K-Ras cell lines. Stimulation of the pathway with growth factors or serum can attenuate phospho-MEK and phospho-ERK induction upon treatment with RAF inhibitors. Additionally, mechanistic studies demonstrate that RAF inhibitors can directly activate RAF kinase activity. Taken together, our data suggests that RAF inhibitors may have opposing functions as both activators and inhibitors of the MAPK pathway, depending on cellular context. These results provide new insight into the therapeutic utility of MAPK pathway inhibitors, and emphasize the importance of correlating target occupancy with pharmacodynamic markers of efficacy, and of targeting defined genetic backgrounds in cancer treatment.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B90.
Abstract B91: RNAi knockdown of Mek2, but not Mek1, increases proliferation of malignant and non-malignant human breast cells
Authors: Jeffress Mara , Beliveau Alain , Campeau Eric , Gray Joe , Yaswen Paul
Published: June 2012

We are using shRNAs against Mek1 and/or Mek2 to determine the individual contributions of Mek1 and Mek2 to proliferation, migration and Mek inhibitor sensitivity in malignant and non-malignant human breast epithelial cell lines. We found in both malignant (MCF7 and MDAMB231) and non-malignant immortalized (184vTERT and MCF10A) breast cell lines that, despite significant shRNA-mediated reductions (>95%) in the expression of Mek1, Mek2, or both kinases, the cells maintained long-term growth. In fact, the shMek2 lines grew significantly faster and were more motile than controls, suggesting that in some breast cancers, drugs that specifically target Mek1 might be more effective than those which target both Mek1 and 2.

In MCF7 cells, levels of phosphorylated (P)-Erk1 and 2 were significantly decreased or undetectable in the presence of Mek shRNAs, indicating that Erk phosphorylation may be dispensable for cell proliferation in these cells. In contrast, in 184vTERT and MCF10A, phosphorylated Erk levels remained constant despite the knockdown of both Mek1 and Mek2. This latter result indicated that, in some breast cells, another kinase is likely to phosphorylate Erk1/2 in the absence of Mek1 and Mek2. The Mek5 kinase is genetically most similar to Mek1 and Mek2, making it an attractive candidate for further investigation. Mek5 is expressed in 184vTERT, and its total protein levels are not altered when Mek1 and 2 are knocked down. A preliminary experiment indicates that siRNA induced knockdown of Mek5 expression leads to decreased ERK1/2 phosphorylation in 184v-TERT cells independently of Mek1/2 expression. We are currently investigating whether this occurs in other breast cell lines.

Collectively, our data indicate that in at least some breast cells, both Mek1 and Mek2 are dispensable for proliferation and survival, and that parallel pathways are sufficient for relaying signals from cell surface receptors, independently of Mek1/2 and/or P-Erk1/2. This data may explain why Mek inhibitors, which target both Mek1 and Mek2, have not done well clinically.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B91.
Abstract B97: Dichloroacetate (DCA) enhances activities of sorafenib against hepatocellular carcinoma (HCC) via modulation of aberrant cellular metabolism of HCC cells
Authors: Shen Ying-Chun , Fan Hsiang-Hsuan , Ou Da-Liang , Liao Sheng-Chieh , Hsu Chiun , Liu Shing-Hwa , Cheng Ann-Lii
Published: June 2012

To meet high energy demand for proliferation, cancer cells rely more on aerobic glycolysis than on oxidative phosphorylation (OXPHOS). This aberrant cellular metabolism of cancer, well-known as the “Warburg effect”, may confer survival advantages and drug resistance to cancer cells. Pyruvate dehydrogenase (PDH) is the gatekeeper of OXPHOS, and is inhibited by pyruvate dehyrogenase kinase (PDK) through phosphorylation. Dichloroacetate (DCA), a synthetic PDK inhibitor, can reverse glycosis to OXPHOS through PDH activation, and has been used for the treatment of congenital lactic acidosis for more than 30 years. The present study explored whether modulating the Warburg effect by DCA can improve the efficacy of sorafenib (a multi-kinase inhibitor of Raf, VEGFR and PDGFR), the current standard systemic therapy for HCC. Modulation of the Warburg effect was evaluated by measurement of lactate in culture medium and PDH activity. In vitro growth-inhibitory effects were measured by median-effect analyses in a panel of human HCC cell-lines (Hep3B, Huh-7, Sk-hep1, HCC36 and HA22T) cultured in normoxic condition. In vivo growth-inhibitory effects were measured in both subcutaneous and orthotopic murine HCC xenograft models. Apoptosis was evaluated by flow cytometry (annexin V staining and subG1 fraction analysis) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Expression of apoptosis-related proteins was analyzed by Western blotting. We found that DCA reduced lactate production and increased PDH activity in HCC cells in a dose-dependent manner, indicating switching glucose metabolism from glycolysis to OXPHOS. DCA (10–60 mM) induced synergistic growth-inhibitory and apoptotic effects with sorafenib in most HCC cell-lines tested. Nude mice carrying subcutaneously or intrahepatically implanted Hep3B cells were treated with vehicle, DCA 100 mg/kg /day (equivalent to the dosage used to treat lactic acidosis in human), sorafenib 10 mg/kg /day, and DCA plus sorafenib, via gavage for 21 days. DCA-sorafenib combination synergistically suppressed the tumor growth without advertently affecting the body weight of mice. Synergistic induction of apoptosis was shown by TUNEL assay. Our data suggest that modulation of aberrant cellular metabolism of HCC cells by DCA may improve the clinical efficacy of sorafenib. (Supported by grants NSC97-3112-B-002-012, NSC98-3112-B-002-007, and NSC98-3112-B-002-037)

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B97.
Abstract B99: Dysregulation of HMGCR and the mevalonate pathway in human cancers
Authors: Penn Linda Z. , Clendening James W. , Pandyra Aleksandra , Boutros Paul C. , Trentin Grace A. , Martirosyan Anna , Trudel Suzanne , Jurisica Igor
Published: June 2012

The importance of cancer metabolism has been appreciated for many years, but the intricacies of how metabolic pathways and oncogenic events overlap remains unclear. By understanding how metabolism contributes to tumorigenesis, we will be able to target these fundamental biochemical pathways and impact patient care.

The mevalonate (MVA) pathway, paced by its rate-limiting enzyme, hydroxymethylglutaryl coenzyme A reductase (HMGCR), is a metabolic pathway required for the generation of a number of fundamental end-products including cholesterol and isoprenoids. Despite being subject to years of extensive research from the perspective of coronary artery disease, the contribution of the MBA pathway to human cancer remains largely unexplored.

Intriguingly, we show that high mRNA levels of 5/6 MVA pathway genes, including HMGCR, correlate with poor prognosis in a meta-analysis of six large expression microarray datasets of primary breast cancer. Indeed, we show that deregulated expression of HMGCR, full-length or splice-variant, increases anchorage-independent growth and tumorigenesis of transformed cells when plated in soft agar or grown as xenografts, respectively. Moreover, we also show that ectopic expression of deregulated HMGCR drives tumorigenesis of non-transformed breast cells a well as normal murine myeloid progenitors. Advancing these studies to an independent tumor-type (multiple-myeloma), shows that loss of the classic feedback response to the blockbuster drugs known as statins, which inhibit HMGCR, further defines dysregulation of the MVA pathway. In addition, we show that this loss of feedback regulation distinguishes the subset of tumor cells that are highly sensitive to statin-induced apoptosis.

Taken together, our results suggest that HMGCR is a candidate metabolic oncogene and provides molecular rationale for further exploring HMGCR inhibitors as anticancer agents.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B99.
Abstract C101: Subcellular distribution study by means of the inherent fluorescence of the investigational drug Triapine and its zinc(II) complex
Authors: Trondl Robert , Kowol Christian R. , Arion Vladimir B. , Jakupec Michael A. , Lichtscheidl Irene , Keppler Bernhard K.
Published: June 2012

The antineoplastic activity of α-N-heterocyclic thiosemicarbazones was discovered several decades ago. Currently the most promising drug candidate of this class of compounds is Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP), which entered several phase I and II clinical trials as an antitumor agent. We discovered that Triapine possesses intrinsic fluorescence properties. This enabled us to monitor for the first time the uptake and intracellular distribution of an α-N-heterocyclic thiosemicarbazone in living human cancer cells by fluorescence microscopy. In addition we synthesized the first zinc(II) complex [Zn(Triapine)Cl2]•HCl of Triapine, which shows fluorescence as well, compared its cytotoxicity in human cancer cells with that of the parental compound and studied the influence of metal complexation on intracellular distribution.

Triapine and its zinc complex were characterized spectroscopically. The UV-Vis spectrum of Triapine in water shows a maximum absorbtion band at 359 nm, whereas the maximum absorbtion of [Zn(Triapine)Cl2]•HCl is slightly shifted to 365 nm. The maxima of the emission spectra of both compounds are at 457 nm when irradiated at ex = 360 nm.

Fluorescence microscopy of living SW480 (colon carcinoma) cells treated with Triapine or [Zn(Triapine)Cl2]•HCl showed that both compounds have a striking affinity to the nuclear membrane and to an organelle structure in the cytoplasm, whereas the localization in the nucleus was strongly different. The zinc complex binds to substructures within the nucleus, which could be verified as nucleoli by immunostaining of the nucleolar protein fibrillarin and co-staining with [Zn(Triapine)Cl2]•HCl. On the contrary, a strong affinity of Triapine to the nucleoli was not detected.

The cytotoxic potencies of Triapine and [Zn(Triapine)Cl2]•HCl were determined in SW480 (colon carcinoma) and 41M (ovarian carcinoma) cells by means of the colorimetric MTT assay. The IC50 value of Triapine is 0.55±0.2 µM in SW480 cells and 0.45±0.03 µM in the 41M cell line. [Zn(Triapine)Cl2]•HCl showed IC50 values of 0.54±0.02 µM in SW480 and 0.52±0.02 µM in 41M cells. Comparing the two substances, no remarkable differences were observed.

Our results demonstrate that Triapine can be visualized with fluorescence microscopy in living cells without any conjugation of fluorophores. Furthermore our findings indicate that metal complexation leads to a different subcellular distribution. Additional studies are ongoing to clearify the influence of metal complexation on the mode of action.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C101.
Abstract C103: In vitro and in vivo anticancer activity of the new ruthenium compound KP1339 against human liver cancer models
Authors: Heffeter Petra , Atil Bihter , Jungwirth Ute , Koerner Wilfried , Micksche Michael , Keppler Bernhard K. , Berger Walter
Published: June 2012

KP1339 is a promising ruthenium based anti-cancer compound. Aim of this study was to test the in vitro and in vivo activity of KP1339 against human liver cancer. As a first step, the cytotoxic potential of KP1339 against a panel of hepatoma cell lines was determined by MTT assay. After 72 h treatment, the mean IC50 value was 124.4 µM with HCC1.2, and HCC3 being most sensitive (IC50 values of 62.9 µM and 67.5 µM, respectively). Notably, the IC50 values did not correlate with intracellular KP1339 levels determined by inductively-coupled plasma mass spectrometry (ICP-MS) measurements. All tested cell lines revealed similar Ru contents (∼5 ng Ru/105 cells) after 1 h drug exposure. To evaluate the mechanisms underlying KP1339 activity, apoptosis induction as well as impact on cell cycle distribution and proliferation of Hep3B cells was evaluated. KP1339 treatment led to typical morphological signs of programmed cell death like cell shrinkage, chromatin condensation and formation of apoptotic bodies. Apoptotic cell death induced by KP1339 was further characterized by PARP cleavage. An increase of cells with mitochondrial membrane depolarization (measured by JC-1 staining and FACS analysis) suggested that at least part of the observed apoptotic cell death is induced via the mitochondrial pathway. Furthermore, KP1339 treatment was found to significantly reduced DNA synthesis rate measured by 3H-thymidine incorporation. With regard to cell cycle distribution, 24 h treatment led to increase of cells in G2/M phase (detected by PI-staining followed by FACS analyses). This was accompanied by strong phosphorylation of the stress kinase P38 and the extracellular signal-regulated kinase (ERK), suggesting that P38 might be involved in delayed apoptosis entry. Currently the activity of KP1339 against several hepatoma cells is evaluated in xenograft experiments using female SCID mice (30 mg/kg i.v.; once a week for two weeks) as compared to the standard therapy Sorafenib (25mg/kg p.o.; five times a week for two weeks). Preliminary results indicate that KP1339 is well tolerated. In the Hep3B model, KP1339 led to a 2.4-fold increase in life span (mean survival of 80 days as compared to 33 days) and thus was superior to Sorafenib monotherapy (1.9-fold survival increase; 60 days as compared to the control). Taken together, KP1339 is a very promising anticancer drug with significant activity against human hepatoma cell lines in vitro and in vivo.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C103.
Abstract C108: A novel mitotic kinesin Eg5 inhibitor exerts the growth-inhibitory effect in cancer cells in a manner independent of induction of monoastral formation but dependent of KRAS mutation status
Authors: Tanaka Toshimitsu
Published: June 2012

Mitotic kinesin Eg5 plays an important role in mitosis, as it is critical for proper bipolar spindle assembly. After the discovery of the first Eg5 inhibitor monastrol, a number of Eg5 inhibitors have been developed as anticancer drugs. We have previously synthesized a series of S-trityl-L-cysteine (STLC) derivatives as Eg5 inhibitors and demonstrated potent inhibitory effect on Eg5 ATPase activity and induction of mitotic arrest with characteristic monoastral spindles in HeLa cells. In this study, we evaluated the growth-inhibitory acitivities of novel STLC derivatives, Compound1, Compound2, and Compound3 by MTT assay in a wide range of human cancer cell lines derived from gastric (MKN1, MKN45, MKN74, NUGC3, NUGC4, NCI-N87), colon (C170, DLD1, HCT15, COLO205), pancreatic (AsPC1, BxPC3, SUIT2) and lung (PC-9, PC-14, H1975, H441, A549) cancers and investigated possible predictive biomarkers. Among the derivatives, Compound3 showed the most potent growth-inhibitory effect and sensitivity between cell lines showed marked differences, with IC50 values ranging from 0.134 to 7.14 M. Pancreatic cancer cell line AsPC1 and non-small cell lung cancer (NSCLC) cell lines H441 and A549 which harbor KRAS mutation showed relatively high IC50 values compared to other cell lines without KRAS mutation. Compound3 induced G2/M arrest in all cell lines as early as 8 hours after the treatment by flowcytometry, while the increase of the apoptotic sub-G1 fraction was not observed in these AsPC1, H441 and A549 cells harboring KRAS mutation. These results suggested that differential sensitivity to Compound3 and attenuated induction of apoptosis by Compound3 were attributed to KRAS mutation. Induction of monoastral spindles formation by Compound3 was observed as early as 8 hours after the treatment not only in sensitive cell lines but in refractory cell lines such as AsPC1 and H441 cells by immunocytochemistry, suggesting the induction of monoastral spindles formation does not work as a predictive biomarker for clinical development. There was no correlation observed between the growth-inhibitory effect and the basal Eg5 expression level of each cell line by western blot analysis. No significant difference was observed in the induction of a spindle checkpoint kinase BubR1 phosphorylation among the cells, a possible predictive biomarker in response to the Eg5 inhibitor treatment by western blot analysis.

Taken together, a novel mitotic kinesin Eg5 inhibitor Compound3 should be considered for further exploration and development and KRAS mutation status was suggested to be a candidate for a predictive biomarker of poor response to Eg5 inhibitors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C108.