bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B85: In vivo radiosensitizer effect of the HDAC inhibitor S78454 on orthotopic human glioblastoma.
Authors: Martinez-Gala Judith , Moyal Elizabeth L. Cohen-Jonathan , Delmas Caroline , Lelievre Helene , Jacquet-Bescond Anne , Kraus-Berthier Laurence , Depil Stephane , Toulas Christine
Published: June 2012

Glioblastoma is an aggressive primary brain tumor with poor outcome, despite a treatment associating surgery and radiotherapy combined with temozolomide. Recent data have shown an in vitro synergy of the HDAC inhibitor S78454 (also called PCI-24781) with ionizing radiation, via inhibition of homologous recombinational (HR) repair of DNA double-strand breaks. We propose in this study to investigate the in vivo radiosensitizer effect of S78454 on human glioblastoma model.

For this, we generated orthotopic U87 human glioblastoma xenografts in nude mice. Intra-peritoneal (i.p.). S 78454 treatment was performed daily starting 10 days after the engraftment. Irradiation was performed in one daily 2 Gy localized fraction during 4 days followed by a second similar 4 days treatment performed after 3 days off (for a total dose of 16 Gy administrated on 11 days). Survival experiments were conducted in mice bearing xenografts in the four different groups of treatment (vehicle, S 78454 alone, radiation alone, S 78454 + radiation). Mice bearing orthotopic xenografts were sacrificed at the onset of neurological signs. Survival curves were then performed.

We first determined the S78454 dosing schedule to obtain a pharmacodynamic effect of the compound in the xenograft. Inhibition of RAD 51 expression, histone H3 and tubulin acetylation in the tumor were obtained 24 hours after daily i.p. treatment with 50mg/kg S78454. These results allowed us to define the schedule treatment combining S78454 with radiotherapy : mice were treated with S78454 50 mg/kg/d i.p., daily from Days 10 to14 and then from Days 17 to 21; one 2 Gy fraction radiotherapy was performed daily from Days 11 to14 and Days 18 to 21.

In these conditions of treatment, S78454 was well tolerated Survival of the mice treated with S78454 alone was not significantly increased while the survival of the mice treated with irradiation alone was significantly increased compared to the control group (P<0.05). In contrast, the survival of the mice treated with the combined treatment was significantly increased compared to the group of animals treated with irradiation alone (P<0.05) and to the group treated with S78454 alone (P<0.01). Effect of S78454 on angiogenesis will be presented. Our results demonstrate that the HDAC inhibitor S78454 radiosensitizes human glioblastoma in vivo and strongly suggest that S78454 may be an interesting drug to combine with radiotherapy in clinical trials for patients with glioblastoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B85.
Abstract B86: Epigenetic regulation of TRAIL mediated by PRAME/EZH2 in cancer.
Authors: Mello Barbara P. , de Carvalho Daniel D. , Campos Antonio HJFM , Andrade Louise DB , Rocha Rafael M. , Binato Renata , Abdelhay Eliana , Carraro Dirce M. , Soares Fernando A. , Amarante-Mendes Gustavo P.
Published: June 2012

TRAIL, a member of the TNF ligand family, was shown to selectively kill oncogenically transformed cells and, therefore, to participate in the cell-mediated immunity against tumors. However, TRAIL is down-regulated in a variety of tumor-bearing patients. On the other hand, PRAME (preferentially expressed antigen of melanoma) is frequently overexpressed in a wide variety of malignant diseases. It was shown that PRAME can function as a repressor of retinoic acid receptor and this transcriptional repression depends on the formation of a complex with a member of the polycomb group, EZH2 (enhancer of zeste homolog 2). Interestingly, TRAIL expression can be positively regulated by retinoic acid. Data from our lab revealed that TRAIL is down-regulated and PRAME is up-regulated during the development of chronic myeloid leukemia (CML) and that their normal levels are restored after complete cytogenetic remission (CCR). Furthermore, there was a significant, negative correlation between the expression of PRAME and TRAIL in CML patients. Overexpression of BCR-ABL in the acute promyelocytic leukemia cell line HL-60 increased the levels of PRAME and decreased the levels of TRAIL. Knocking-down of either PRAME or EZH2 in K562 CML cell line resulted in a dramatic up-regulation of TRAIL. We concluded that PRAME up-regulation by BCR-ABL plays an important role in CML pathogenesis by repressing retinoic acid signaling pathway and, consequently, down-regulating TRAIL expression, a potent anti-cancer agent as an inducer of the extrinsic pathway of apoptosis. On the basis of these results, we performed bioinformatics analysis, using the Oncomine Research platform, for PRAME and TRAIL expression on solid tumors. We found several works in which PRAME and TRAIL expressions were negatively correlated. In particular, PRAME was up-regulated while TRAIL was down-regulated in kidney, lung, prostate and sarcoma compared to normal tissues. These expression patterns of PRAME and TRAIL were confirmed by real-time PCR in tumor cell lines of prostate, breast, colon and pharynx. At this moment, we are expanding the validation of bioinformatics data in a larger number of tumor cell lines as well as in tumor and normal tissues from patients with different forms of cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B86.
Abstract B93: Gene fusions detected by whole transcriptome sequencing of lung adenocarcinoma.
Authors: Kohno Takashi , Ichikawa Hitoshi , Totoki Yasushi , Yasuda Kazuki , Hiramoto Masaki , Nammo Takao , Sakamoto Hiromi , Tsuta Kohji , Furuta Koh , Shimada Yoko , Iwakawa Reika , Chiku Suenori , Yamanaka Itaru , Watanabe Shunichi , Sekine Ikuo , Ogawa Seishi , Tsuda Hitoshi , Yoshida Teruhiko , Yokota Jun , Shibata Tatsuhiro
Published: June 2012

A considerable proportion of lung adenocarcinoma (LADC) cases develop through activation of oncogenes, i.e., somatic mutations in either the EGFR (10–40%) or KRAS (10–20%) genes, or fusion of the ALK gene (5%) with the EML4 or KIF5B genes, in a mutually exclusive manner. Tyrosine kinase inhibitors that target EGFR and ALK proteins are particularly effective for treating LADCs carrying EGFR mutations and ALK fusions, respectively. In this study, whole transcriptome sequencing (RNA sequencing) of 30 Japanese LADCs and three adjacent non-cancerous lung tissues was performed to identify novel chimeric fusion transcripts that could be potential targets for therapy. Analysis of more than 2 × 107 paired-end reads obtained by RNA sequencing and subsequent validation by Sanger sequencing of the reverse transcription (RT)-PCR products identified EML4-ALK fusions in two cases and a few other gene fusions in each single case. The two EML4-ALK positive LADC cases were negative for EGFR and KRAS mutations and also negative for other gene fusions. Therefore, the authenticity of ALK fusions as a driver mutation was validated. The prevalence and oncogenic property of novel gene fusions identified are being studied.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B93.
Abstract C110: Mutation and chemosensitivity profile of a panel of 25 patient-derived melanoma xenografts.
Authors: Fiebig Heinz Herbert , Vuaroqueaux Vincent , Maier Armin , Kelter Gerhard , Schueler Julia B. , Krumbach Rebekka
Published: June 2012

Melanomas account for a notable proportion of cancers worldwide. The heterogeneity and biological characteristics of melanomas present unique therapeutic challenges. Recently a number of somatic mutations were determined, which influence the prognosis and thus therapeutic needs markedly. Here we present a panel of patient-derived melanoma xenografts, established by Oncotest from primary patient material. From 80 patient explants, growth in serial passage was observed in 43% of the cases. Molecular profiling included mutational analysis for BRAF, PI3Kalpha, KRAS, NRAS, TP53, CTNNB1, MET and JAK3 using OncoCarta™ and exon sequencing. The chemosensitivity profile was determined in vitro using 2D and 3D culture assays.

Clinically relevant BRAF mutations were the most prevalent mutation and were found in almost two thirds of the melanoma models. The second most common gene mutated was NRAS, with about one quarter of the models harbouring activating mutations of NRAS. PI3Kalpha mutations were rare, and for KRAS no mutations were detected. Most of the melanomas showed multiple mutations of the investigated genes. Chemosensitivity profiling included cytotoxic (e.g. cisplatin, carboplatin, vinblastine, vincristine, ifosfamide, paclitaxel, and docetaxel), as well as targeted drugs (e.g. sorafenib and PLX-4720). The compounds showed diverse patterns of selectivity and potency: Differential activity was determined e.g. for vinblastine, vincristine, paclitaxel, and docetaxel, but also for the BRAF inhibitor PLX-4720. PLX-4720 was most active in melanoma models carrying the BRAF mutation as determined in monolayer and soft agar assays. Activity of vincristine revealed a correlation of sensitivity with a BRAF wild type genotype and resistance with BRAF mutation. The latter result was also described recently for melanoma patients with BRAF mutations, showing a diminished duration of response to treatment with vincristine, dacarbazine, bleomycin, lomustine, and human leukocyte interferon.

A collection of 25 patient-derived melanoma models is presented. Their mutational status as well as their responsiveness towards many cytotoxic and targeted agents was analyzed. This extensive genotypic and phenotypic characterization makes them a valuable tool for hypothesis generation and testing.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C110.
Abstract C113: Modeling cetuximab clinical trial by conducting in vivo efficacy evaluation in large panel CRC patient-derived xenograft (PDX) models.
Authors: Chen Dawei , Yang Mengmeng , Deng Jiangyun , Song Xiaoming , Cai Jie , Chen Taiping , Chen Yiyou , Li Henry
Published: June 2012

Patient derived xenograft (PDX) models without in vitro manipulation mirror patients' histopathological and genetic profiles1,2. This has the potential to significantly improve the predictive power of preclinical cancer animal models, and more importantly to enable the identification of predictive biomarkers for targeted therapeutics.

In this study, we set out to mimic a small size clinical trial by testing cetuximab in a large cohort of CRC models. Among 19 CRC models tested, 7 models were found sensitive (37%) and 12 resistant (63%) to cetuximab. All 3 models with BRAF activating mutation are found resistant to cetuximab. Of the 11 models with KRAS activating mutation, the response profile is more complex, with 6 models resistant and 5 models responsive to cetuximab. It is interesting that 3 out of the 4 models with G13D mutation are sensitive to cetuximab. Of the 5 models which harbor neither KRAS nor BRAF mutation, 3 models are resistant, suggesting that other oncogenic drivers are involved. Interestingly, one of the resistant models harbors an EGFR R776H activating mutation.

The observation that KRAS mutations such as G13D and G12C are still sensitive to cetuximab, although contrary to the accepted dogma that CRC patient with mutant KRAS does not benefit from cetuximab3, is consistent with the recent findings in clinic4–7. Other KRAS mutations such as A146T and R61H were found in the resistant models, may imply that mutations besides codon 12 and 13 can play roles in the pathogenesis of CRC and the drug resistance. The overall resistant profile of models with BRAF mutation may also suggests another potential risk factor which warrants further investigation.

In summary, our results have demonstrated that large panel HuPrime® can be used to effectively evaluate investigational new drugs for efficacy prediction and to discover predictive biomarkers to guide patient stratification for clinical development.

References

1. E. Marangoni et al., Clin Cancer Res 13, 3989, 2007.

2. L. Ding et al., Nature 464, 999, 2010.

3. A. Lievre et al., Cancer Res 66, 3992, 2006.

4. W. De Roock, V. De Vriendt, N. Normanno, F. Ciardiello, S. Tejpar, Lancet Oncol 12, 594, 2011.

5. W. De Roock et al., JAMA 304, 1812, 2010.

6. W. De Roock et al., Lancet Oncol 11, 753, 2010.

7. R. Kirk, Nat Rev Clin Oncol 8, 1 2011.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C113.
Abstract C120: Quantitative analysis of ERK phosphorylation by single cell phospho-specific flow cytometry in colon cancer cells as an input for pharmacokinetic/pharmacodynamic modeling of U0126 concentration.
Authors: Hwang Jee Won , Ahn Joong Bae , Rha Sun Young , Roh Jae Kyung , Chung Hyun Cheol , Bae Soo Kyung , Bae Soo Hyeon , Shin Sang Joon
Published: June 2012

Background: To evaluate intracellular levels of phosphorylated proteins in many signaling pathways, Western blot, immunohistochemical and immufluorescence have been generally used until now. However, to establish pharmacokinetics-pharmacodynamics modeling of various drugs including antitumor agents, precise quantitative measurements of intracellular level of phosphorylated signaling molecules are critically needed. Methods and Materials: In this study, we investigated the ability of single cell phospho-specific flow cytometry to detect dynamic change of intracellular level of phosphorylated-ERK (pERK) as a pharmacodynamic marker of MEK/ERK inhibitor U0126 in human colon cancer cell lines. We also measured the intracellular concentration of U0126 in the cell lines using High-performance liquid chromatography.

Results: The fluorescence intensity changes by pERK-specific single cell flow cytometry after U0126 treatment was corresponded to the pattern of results obtained by Western blot and immunofluorescence staining in dose- and time-dependent manner. In HCT116 and HT-29 human colon cancer cells, the basal fluorescence intensity of pERK was inhibited with a 0.2- to-0.8 fold decrease according to increasing concentration of U0126 (0.1 − 100 uM). We detected the rapid decrease of pERK activity within 15 min after U0126 treatment; this decrease was continued to 2 hours. And we observed the gradual recovery of pERK level closely basal status until 24 hours. The concentration of U0126 in the media was associated with decreased activity of pERK but the intracellular concentration of U0126 was not.

Conclusion: Our data obtained by single cell phospho-specific flow cytometry are more sensitive, rapid, and quantitative in parallel compared with western blot. This accuracy allowed us to acquire precise inputs in order to establish pharmacokinetics-pharmacodynamics modeling of various anti-tumor agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C120.
Abstract C121: A phase 1 QTc study of tivozanib in patients with advanced solid tumors.
Authors: Chadha Manpreet , Infante Jeffrey R. , Cotreau Monette M. , Jacobson Lindsey , Strahs Andrew L. , Slichenmyer William , Vargo Dennis L. , Moore Kathleen
Published: June 2012

Background: Tivozanib is a potent, selective, long half-life tyrosine kinase inhibitor of vascular endothelial growth factor receptors-1, -2, and -3 that is currently being tested in a Phase III study in patients with renal cell carcinoma and Phase I/II studies of other solid tumors. Previous preclinical and clinical studies have shown that tivozanib displays strong antiangiogenic and antitumor effects. Preclinical and retrospective electrocardiogram (ECG) analyses did not suggest an effect of tivozanib on QTc, although this has not been prospectively assessed according to ICH E14 Cardiac Assessment of New Drugs Guidelines. This open-label, non-randomized, single-arm study prospectively investigated the proarrhythmogenic potential of tivozanib on the QTcF interval and its morphology on the ECG and ECG-pharmacokinetic (PK) relationship in patients with advanced solid tumors.

Methods: Patients with advanced solid tumors, an ECOG score ≤1 and life expectancy ≥3 months were eligible. Patients received 1.5 mg of tivozanib orally, once daily for 21 days. Patients with a repeated baseline QTcF >480 ms were excluded. Serial blood samples and time-matched, triplicate, 12-lead ECGs were collected on: Day 1 20–30 minutes pre-dose (no blood sample collected), immediately pre-dose, and at 2.5, 4, 5, 6, 8, and 10 hours post dose; Day 2 pre-dose evaluation was taken approximately 24 hours post Day 1 dose; Day 8 (±1 day) pre-dose, and at 2.5, 5, and 8 hours post dose; Day 21 pre-dose and at 2.5, 4, 5, 6, 8, and 10 hours post dose; and Day 22 at approximately 24 hours post Day 21 dose. Additional safety parameters were evaluated by assessment of clinical laboratory tests, physical examinations, vital signs, and recording of adverse events.

Results: Fifty patients with advanced solid tumors (males, 17; median age, 63 years; 94% white) who received at least one dose of tivozanib were evaluable for the present study. Preliminary adverse event data showed that there were no clinically significant changes in QTcF from baseline. Further analysis will be completed, and final safety and ECG-PK modeling will be presented.

Conclusions: Preliminary data suggest that tivozanib at 1.5 mg daily over a 21-day period does not cause clinically significant prolongation of QT/QTc over baseline. Data from this study suggest that the safety and PK profile of tivozanib was similar to that observed in previous studies, and that the findings are consistent with the ECG evaluation in a monkey telemetry study.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C121.
Abstract C132: Radiosensitization of solid tumors by an MDM2 inhibitor (HLI373989).
Authors: Cerna David , Carter Donna , Yoo Stephen S.
Published: June 2012

The tumor suppressor p53 is a key molecule in the prevention of cancer. More than half of all human cancers have mutation or loss of the p53 gene. A significant percentage of the other half contain overexpression of Hdm2 which serves as a ubiquitin ligase (E3) to target p53 for degradation. While it has been established that an inhibitor of Hdm2 ubiquitin E3 ligase activity HLI373 (5-(3-dimethylaminopropylamino)-3,10-dimethyl-10H-pyrimido(4,5-b)quinoline-2,4-dione, NSC373989), selectively kills tumor cells harboring wild-type p53, we investigated the effect of the Hdm2 inhibitor HLI373, in the response to ionizing radiation in solid tumor lines expressing wild-type, mutant or null p53 in vitro and in vivo. We now report HLI373 enhances radiosensitivity in a panel of human tumor cell lines in p53 deficient and competent cell lines using clonogenic assay. HCT116 cells exposed to HLI373 (5–7.5 uM) for 16 hours before and maintained in the medium after irradiation had an increase in radiosensitivity. Cell cycle distribution of treated cells after 16 hours was measured using flow cytometry. In a comparison of wildtype and null cell lines, cell cycle arrest in G1 was only detected in cells with active p53 at 5uM of HLI373. In contrast, cells with a null or mutated p53 progressed to and arrested in G2. In addition, wild type p53 cells pretreated with 5 uM HLI373 and irradiated continued to be arrested in G1 and did not progress to a G2. However, cells without p53 treated with HLI373 and radiation increased in accumulation in G2. Treatment with higher dosages of HLI373 (>10uM) resulted in the accumulation of cells in G2 regardless of p53 status. These results indicate that HLI373 enhances tumor cell radiosensitivity in vitro and agents such as HLI373 may have potential for use in the clinics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C132.
Abstract C141: Screening of novel RNA aptamer(s) that selectively bind(s) to prostatic acid phosphatase.
Authors: Kong Hoon Young , Byun Jonghoe
Published: June 2012

Prostate cancer is becoming one of the most popular non-skin cancers in Korea due to continued increase of western life styles. Since earlier detection of prostate cancer can lead to complete cure, a lot of studies have been done on prostate cancer markers such as prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP) and prostate stem cell antigen (PSCA). Among these biomarkers, PAP is gaining renewed interest because of its potential merits in diagnosis and therapeutics. PAP, which was initially identified in 1935, is an abundant enzyme that is synthesized in prostate epithelial cells and has enzymatic activity in acidic conditions. PAP expression is androgen-independent and increases in proportion to prostate cancer progression. Indeed, PAP expression level has high correlation with cause-specific survival and its dephosphorylation activity is believed to play an important role in the incidence and development of prostate cancer. Here, we screened the in vitro transcribed RNA libraries to get aptamer(s) that can selectively bind to PAP. Aptamers are oligonucleic acid molecules that bind to a specific target through formation of tertiary structures. These chemical antibodies can bind their target molecules with a high affinity and specificity and have the advantage of being less immunogenic in vivo. For in vitro selection, the ectracellular domain of PAP were cloned from PC3 cells and subjected to 6 rounds of SELEX procedure. This resulted in enrichment of the RNA aptamers that bound to the PAP ectodomain with 3-fold higher affinity than the library control as evidenced by real-time RT-PCR and gel shift assay. Sequence analysis of the aptamers revealed three distinct groups (G1, G2, N) having variable sequences within the region comprising nucleotides 43 to 87. The Kd values of the best clones with the highest affinity were within 120nM. These clones could also bind to the PAP-positive cells such as PC3 and LNCaP cells. Interestingly, 2'-OH-modification of the aptamers led to loss of binding, implying that 2'-F modification may be essential in binding to PAP. Characterization of these aptamers is currently underway and initial minimization results suggest that the structure coming from the 50 nucleotides is essential for binding to PAP. With further optimization, these PAP RNA aptamers could find wide application in theragonosis of prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C141.
Abstract C145: Compounds that inhibit the activation of Grb2 by EGFR, HER2, and HER3 complexes.
Authors: Story Michael J. , Marshall Philip A. , Ayoub Mohammed A. , Pfleger Kevin DG
Published: June 2012

Purpose: To demonstrate that ORIL003 (Glc4-Rha2-Glc-diosgenin; full structure will be disclosed) inhibits the activation of Grb2 by EGFR, HER2 and HER3 complexes using the Receptor-Heteromer Investigation Technology (Receptor-HIT) on the bioluminescence resonance energy transfer (BRET) platform.

Introduction: The unique property of inhibiting a range of receptor tyrosine kinases (RTKs) in the cell plasma membrane has been demonstrated in a family of steroid saponins. As the compounds have a pan-inhibitory effect, they are effective in inhibiting a wide range of cancer cell types, including lung, breast, prostate, colorectal, melanoma, ovarian and leukemia.

Methods: Receptor-HIT, previously published in the form of G protein-coupled receptor heteromer identification technology (GPCR-HIT; See et al, 2011 Assay Drug Dev Technol 9:21–30), was used to determine close proximity and activation of ErbB growth factor receptors and Grb2. EGFR, HER2 and HER3 were tagged with Rluc8 (a variant of Renilla luciferase) as donor; Grb2 was tagged with GFP2 (a variant of green fluorescent protein) as acceptor. Human embryonic kidney 293 cells were stimulated with either epidermal growth factor (EGF) or heregulin (HRG) at a concentration of 20–50 nM. ORIL003 (as an example of the steroid saponin family) was applied to the cells at a concentration of 10 μM, which was known from in vitro studies to completely inhibit cancer cell growth.

Results: Stimulation by EGFR or HRG resulted in recruitment of Grb2. This recruitment was inhibited by the use of ORIL003 in a dose-dependent manner. Furthermore, constitutive interactions between the ErbB growth factor receptors and Grb2 also appear to be inhibited by ORIL003.

Conclusions: ORIL003 is effective in inhibiting the activation of Grb2 by EGFR, HER2 and HER3 complexes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C145.
Abstract C150: CaMKKβ-mediated phosphorylation of Beclin 1 regulates autophagy and cell differentiation in neuroblastoma cells.
Authors: Wu Xiao-Qi , Li Dan-Dan , Deng Rong , Jiang Shan , Feng Gong-Kan , Zhu Xiao-Feng
Published: June 2012

Autophagy is an evolutionarily conserved degradative pathway by which components of the cells are degraded in the lysosome in response to stress. Beclin 1 plays a central role in autophagy. Pioneering work has indicated that the increase in cytosolic Ca(2+) can induce autophagy via the CaMKKβ-mediated activation of AMPK. Here, we demonstrate a novel signaling pathway in which CaMKKβ regulates autophagy and differentiation. We found that Ca(2+) mobilizing agents (ionomycin and EB1089) induced phosphorylation of Beclin 1 and autophagy through CaMKKβ-dependent pathway. Using In vitro kinase assay we demonstrated that CaMKKβ directly phosphorylated Beclin 1 Ser90. Moreover, autophagy induced by ionomycin or EB1089 contributed to degradation of Id1 (inhibitor of differentiation). The inhibitors of autophagy including Bafilomycin A1 and chloroquine and RNA interference of autophagy-associated gene atg 5 markedly increased the protein levels of Id1 and abrogated neuroblastoma cell differentiation. Taken together, CaMKKβ promoted autophagy by directly phosphorylating Beclin 1 at Ser90, and thus induced cell differentiation in neuroblastoma cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C150.
Abstract C177: Chemical-driven biological probing: Discovery of a novel antimetastatic drug.
Authors: Xu Li , Farmer Rebecca , Huang Xiaoke , Valsecchi Matias , Pavese Janet , Voll Eric , Biddle Margaret , Nibbs Antoinette , Pruell Sean , Avram Michael , Scheidt Karl , Bergan Raymond C.
Published: June 2012

Chemicals constitute richly informative probes, have high resolving potential and are able to uncover complex biological processes. Harnessing this potential provides an avenue for the discovery of new therapeutics that act via novel mechanisms. We focused these principles upon an intractable problem: cancer metastasis. We started with a chemical scaffold with broad bioactivity, with desirable drug-like properties and that would support synthetic diversification. Initially focusing upon human prostate cancer (PCa) we then coupled fragment-diversification and novel synthetic routes to upfront positive selection screens (inhibition of cell motility) and negative selection screens (cell toxicity) in an iterative fashion. We thereby efficiently synthesized a new class of bioactive compounds that inhibits systemic PCa metastasis at low nanomolar concentrations. Efficacy against other cancer types was demonstrated. Extensive investigations indicate high specificity and no toxicity. Target validation studies point to inhibition of protein-protein interaction motifs. Together, these studies support the notion that this approach is powerful, can be broadly applied across biological systems, and constitutes a paradigm. Specifically, they have led to the discovery of a novel acting drug that inhibits human cancer metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C177.
Abstract C199: Sorafenib targets BCR kinases and blocks migratory and microenvironmental survival signals in CLL cells.
Authors: Lopez-Guerra Monica , Xargay-Torrent Silvia , Perez-Galan Patricia , Saborit-Villarroya Ifigenia , Rosich Laia , Villamor Neus , Aymerich Marta , Roue Gael , Campo Elias , Montserrat Emili , Colomer Dolors
Published: June 2012

Targeting deregulated kinases has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL). Here, we report that the multikinase inhibitor sorafenib selectively induces apoptosis in the majority of CLL cases, with preference for the IGVH unmutated cases and for lymph node-derived CLL cells. We also show that ERK inhibition by sorafenib in CLL cells is an early but not sustained event, thus downregulation of RAF/MEK/ERK pathway is not a crucial effect of this agent in CLL. Among all the different kinases modulated by sorafenib in CLL cells, the blockage of BCR-associated tyrosine kinases, SRC and SYK, is particularly relevant for its action. In line with this, we demonstrate a strong synergy when combining sorafenib with two pharmacological BCR inhibitors, R406 and dasatinib. Importantly, sorafenib overcomes BCR stimulation as well as the protective effect of the stromal coculture. This compound resensitizes CLL cells cultured with stroma to fludarabine- and bendamustine-induced apoptosis. Furthermore, sorafenib inhibits CLL migration toward CXCL12 and blocks both CXCL12- and BCR-induced FAK activation. All these results indicate that sorafenib could overcome the tumoral microenvironment and might be particularly effective in BCR signaling inhibition in CLL cells with higher migratory potential, especially in high risk patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C199.
Abstract C2: Selection for combinational studies through biomarker stratification in colorectal patient-derived explant models.
Authors: Alferez Denis G. , Dry Jonathan R. , Holt Sarah V. , Weston Susie L. , McWalter Gael , Beran Garry J. , Revill Mitch , Smith Paul D. , Guichard Sylvie , Wedge Stephen R. , Hedge Phillip , Wilkinson Robert W.
Published: June 2012

Patient-derived explant models (PTX) show a higher genetic and pathological heterogeneity than cell line derived xenograft models, and may have greater clinical relevance in cancer drug development and personalised oncology treatments than cell line derived xenograft models. Therefore PTX models can potentially be used to provide additional confidence in patient stratification hypotheses for novel therapies or combination approaches.

To examine comparative pharmacological responses in PTX models, the monotherapy activity of the MEK inhibitor selumetinib was determined in colorectal cancer PTX models that were stratified according to a MEK pathway “functional activation” mRNA signature, originally derived using a broad human tumor cell line panel (1). Tumor passage, immunohistochemical and genetic markers of target pathway activity were also assessed to collectively select 6 models of high interest for examining response to treatment. In addition, to monotherapy treatment, 3 models were also used to examine the combination of selumetinib with the mTORC1/2 kinase inhibitor AZD8055.

The results suggest that the MEK pathway mRNA signature is applicable in colorectal PTX models and enables selumetinib responsive versus non-responsive models to be identified. Combination therapy of selumetinib and AZD8055 has also shown additional therapeutic benefit, similar to that observed in selected cell line derived xenografts.

These data illustrate that use of PTX panels can be applied to substantiate in-vitro derived biomarker-led hypotheses for a given therapeutic approach. In addition, biomarker stratification has focused experimental design to test novel:novel combinations and may enable stratification for prospective patient subsets. Collectively this approach to disease modelling could significantly reduce wide screening in vivo studies and steer more refined clinically relevant hypotheses, whilst allowing for a reduction in animal usage.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C2.
Abstract C215: Depletion of the resistance-associated βIII tubulin isotype enhances eribulin's potency in suppressing microtubule dynamic instability and promotes inhibition of both microtubule growth and shortening.
Authors: Lopus Manu , Miller Herbert , Azarenko Olga , Joseph Silja , Nomoto Kenichi , Wilson Leslie , Jordan Mary Ann
Published: June 2012

Eribulin mesylate (Halaven®) is a synthetic analog of the microtubule-targeted natural product halichondrin B, originally isolated from the marine sponge, Halichondria okadai. Recently, the US FDA approved eribulin mesylate for the treatment of patients with metastatic breast cancer who have previously received at least two chemotherapeutic regimens for the treatment of metastatic disease. Prior therapy should have included an anthracycline and a taxane in either the adjuvant or metastatic setting. Seven β-tubulin isotypes have been identified, and they are differentially expressed in a variety of tissues and tumors. A high level of βIII-tubulin often correlates with poor clinical outcome in several human tumors and with resistance to microtubule-targeted agents in certain human cancer cell lines and tumors. Here, we report the strong preferential suppression by eribulin of microtubule dynamic instability of purified microtubules depleted of βIII-tubulin in vitro. Tubulin was purified from bovine brain by cycles of temperature-dependent assembly and disassembly followed by phosphocellulose chromatography, and depleted of III-tubulin by immunoaffinity chromatography. Depletion of βIII-tubulin did not affect the inherent dynamic instability of the microtubules. Eribulin (100 nM) suppressed dynamic instability of βIII-tubulin-depleted microtubules significantly more strongly than it suppressed dynamic instability of microtubules assembled from unfractionated tubulin. Specifically, with microtubules composed of unfractionated tubulin, eribulin significantly suppressed only the growth rate (by 27%) and dynamicity (by 36%). The shortening rate and the catastrophe and rescue frequencies were suppressed only insignificantly (by 14%, 17%, and −2%, respectively). However, with βIII-tubulin-depleted microtubules, eribulin significantly suppressed almost all dynamic instability parameters including the growth rate (40%), shortening rate (42%), and the catastrophe and rescue frequencies (by 49% and 33%, respectively) leading to an overall suppression of microtubule dynamicity of 58%. Determination of the binding of eribulin to βIII- tubulin-depleted microtubules and to unfractionated microtubules is in progress. Acknowledgment: Supported by a grant from Eisai Inc.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C215.
Abstract C222: Diverting Hsp70 trafficking as a tool for stimulating anticancer immunity.
Authors: Juhasz Kata , Thuenauer Roland , Spachinger Andrea , Duda Erno , Horvath Ibolya , Vigh Laszlo , Sonnleitner Alois , Balogi Zsolt
Published: June 2012

The stress-inducible heat shock protein 70 (Hsp70) is normally destined for cytosolic localization. In tumors, Hsp70 is frequently localized to the cell surface and released from cells, unusual features that stimulate the immune system against cancer cells. Interestingly, it has been shown that overexpression of Hsp70 enhances the immunogenicity of tumor cells in animal models. Here we investigated potential mechanisms by which overexpression of Hsp70 in B16 melanoma cells may increase surface expression and release. Flow cytometry measurements revealed an increased cell membrane concentration upon elevated level of intracellular Hsp70. Hsp70 was detectable in subcellular fractions enriched in endosomes/ lysosomes. An accumulation of excess Hsp70 was seen in high Hsp70 expressing cells, in particular in the purified lyosomal fractions. In low expressing cells apparent trafficking of Hsp70 was confined to the endosomal system. In contrast, high intracellular Hsp70 concentration was associated with a predominant lysosomal routing. Remarkable changes were observed in Hsp70 trafficking upon heat stress, a common approach used to facilitate Hsp70 release. In control cells a dramatic depletion of surface-exposed Hsp70 was paralleled with increased endosomal recycling of internalized protein. In contrast, excess intracellular Hsp70 prevented the membrane transport from heat-induced depletion, and allowed transport of surface-exposed Hsp70 to lysosomes secreting their cargo in a soluble form. Therefore excess Hsp70 appears to be decisive in terms of both trafficking to the plasma membrane and stress-induced active release. Altogether, our findings demonstrate a vesicular trafficking system bringing excess intracellular Hsp70 to fulfill its immunostimulatory role.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C222.
Abstract C29: Elevated CDKN1A (p21) expression in human cancer cell variants selected for resistance to the novel microtubule depolymerizing agent BAL27862 (active moiety of BAL101553).
Authors: Duran George E. , Lane Heidi , Bachmann Felix , Sikic Branimir I.
Published: June 2012

BAL27862 is a synthetic small molecule that induces tumor cell apoptosis due to inhibition of tubulin polymerization via a potentially new mechanism of action. Administered as the prodrug BAL101553, BAL27862 is currently undergoing Phase I evaluation in advanced cancer patients. We have previously demonstrated that BAL27862 retains activity in a broad panel of MDR1(+) and MDR1(−) human breast and ovarian cancer cell variants selected for resistance to taxanes (Abstract 4412, AACR 2010). In this current study, we established five variants selected for stable drug resistance in cell lines from human lung cancer (H460 and A549), acute T-cell leukemia (Jurkat), ovarian cancer (SK-OV-3) and cervical cancer (HeLa). These variants are approximately 3- to 24-fold resistant to BAL27862. The resistance is not transporter mediated, with no activation of ABCB1 (MDR1/P-glycoprotein) following drug selection, no change in activity in cells co-incubated with known P-glycoprotein inhibitors (PSC-833, 2 μM) and no difference in either accumulation or retention of [14C]-BAL27862 relative to parental controls. Tubulin polymerization assays indicated a higher proportion of tubulin in the polymerized fraction from resistant lines, indicating microtubule stabilization. Consistently, most lines exhibited increased sensitivity to microtubule stabilizers (e.g. resistance factors [RF = antiproliferative IC50 resistant line/parental line] for paclitaxel: H460=0.4; Jurkat=0.7). Strikingly, differential responses to standard microtubule-destabilizing agents were evident. For example, RFs for Jurkat cells were lower for both vincristine (2.5) and colchicine (10) as compared to BAL27862 (24); whereas, similar RFs were measured in SK-OV-3 (3, 5 and 4, respectively). Finally, relative to parental controls, altered tubulin isoform content was observed in some BAL27862-selected lines, but elevated CDKN1A (p21) protein expression was observed in 4 out of 5 of the resistant variants (SK-OV-3, H460, A549 and HeLa). Q-PCR analysis indicated an associated elevation in p21 mRNA levels only in lines with enhanced protein expression. As aberrant p21 expression has been previously related to poor prognosis and chemotherapy resistance in cancer patients, the potential of using this biomarker to support patient stratification efforts in future trials is worthy of further study. The functional significance of p21 to the resistance phenotype, and other key alterations identified through more detailed genomic profiling (Stanford's HEEBO microarrays), will be studied by gene transfer and gene silencing experiments.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C29.
Abstract C3: Validation and fitness testing of a quantitative immunoassay for HIF1α in biopsy specimens.
Authors: Kinders Robert J. , Park Sook Ryun , Khin Sonny , Hollingshead Melinda G. , Parchment Ralph E. , Tomaszewski Joseph E. , Doroshow James H.
Published: June 2012

HIF1α is an important marker of hypoxia in human tumors that is altered in a number of cancers, but a reproducible method to determine changes in HIF1α protein expression in human tumor biopsy specimens has not been available. HIF1α is being used as a pharmacodynamic marker in a clinical trial of metronomic Topotecan (CTEP# 8610, 9534). However, an important limitation for studying the response of HIF1α to cancer therapeutic agents is the lability of the protein, in the presence of oxygen, upon collection of the sample. We have devised a method of specimen collection, handling and extraction that preserves and stabilizes HIF1α levels in tumor biopsies. Employing this specimen handling method allowed validation of a two-site immunoassay for HIF1α quantitation in solid tissue extracts, such as tumor biopsies. Intra-assay variability was less than 10% and inter-assay variability was less than 20%. Accuracy, assessed by spike recovery, was 100 +/− 10%. HIF1α readings declined linearly with decreasing sample load over a range of 1 to 10 μg protein per well. HIF1α is being used as a pharmacodynamic marker in a clinical trial of metronomic Topotecan, so fitness for purpose was demonstrated by quantifying a reduction in HIF1α protein levels following topotecan treatment of a xenograft model. HIF1α was also demonstrated to be upregulated under low oxygen tension culture conditions in DU145 human prostate cancer cells. The HIF1α immunoassay is currently being transferred to the NCI's National Clinical Target Validation Laboratory for use in support of NCI-sponsored early clinical trials. This Research has been funded with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C3.
Abstract C41: Analysis of Her-2/neu status in brain metastases of breast cancer.
Authors: Rotin Daniil
Published: June 2012

Introduction: Breast carcinoma is the second common type of cancer metastasizing into the brain. Although the frequency of Her-2 positive primary breast tumors is no more than 30%, a higher occurrence of brain metastases has been reported for patients with positive Her-2/neu primary tumors. We investigated here expression of Her-2 protein and amplification of Her-2 gene in brain metastasis tissues of breast carcinoma patients.

Patients and Methods: FFPE tumor tissues were obtained from consented patients treated with surgery at the NN Burdenko Neurosurgery Institute in 2004–2007. Total of 42 cases of brain metastases (BM) with breast tissue origin were evaluated. Solitary BM occurred in 59% (16/27) patients, while multiple metastatic lesions were observed in 41% (11/27) patients. The median age was 53 (range 29 − 73 years). Her-2/neu gene amplification was analyzed by fluorescent in situ hybridization (FISH). Levels of protein expression were detected by immunohistochemistry (IHC) according to the DAKO HercepTest grading system. Membranous staining intensiveness was estimated by a semi-quantitative method (from 0 to 3+). Cases with Her-2 IHC expression score of 2+ were reconfirmed by FISH analysis. Statistical analysis was done using STATISTICA 6.0 software.

Results: Over-expression of Her-2 protein was observed in 50% (21/42) cases, while only in 7% (3/42) cases level of HER-2 protein was graded as negative (scores 0 and 1+). In 43% (18/42) cases IHC score was scored as 2+, and for those cases HER-2/neu gene amplification was performed using FISH analysis. Amplification of Her-2/neu was detected in 72% (13/18) cases in that group. Over-expression of Her-2/neu was similar in tissues from patients with a single metastatic lesion (63% (10/16) cases) compared to that of patients with multiple brain metastases (60% (25/42) of cases).

Conclusions: Despite relatively lower incidence in primary tumors, Her-2/neu positive breast cancer tumors prevail in breast cancer brain metastases (about 60%), perhaps attributing to the higher malignant potential of such tumors. We found no difference in Her-2/neu protein expression levels in patients with single and multiple brain metastases.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C41.