bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3679: Inhibition of LSD1 disrupts global EWS/ETS transcriptional function in Ewing sarcoma
Authors: Theisen Emily Rose , Sankar Savita , Bearss Jared , Mulvihill Timothy , Sorna Venkataswamy , Sharma Sunil , Lessnick Stephen L.
Published: May 2015

Ewing sarcoma is an aggressive pediatric bone tumor characterized by an absolute reliance on the transcriptional activity of the EWS/ETS family of transcription factor fusion oncoproteins. The most common fusion is EWS/FLI, present in 85-95% of cases, though less common fusions include EWS/ERG, EWS/ETV1, EWS/ETV4 and EWS/FEV. EWS/FLI utilizes lysine-specific demethylase 1 (LSD1) as member of the NuRD complex to mediate transcriptional repression of critical tumor suppressors in Ewing sarcoma. LSD1 overexpression has been observed in clinical samples from Ewing sarcoma patients and small molecule blockade of LSD1 using tranylcypromine has been suggested as a therapeutic approach for Ewing sarcoma. We therefore evaluated the effects of LSD1 inhibition with the small molecule inhibitor, HCI-2509, in both in vitro and in vivo models of Ewing sarcoma. HCI-2509 is an LSD1 inhibitor with a Ki of ∼30 nM and multiple Ewing sarcoma cell lines show IC50s in cell viability assays from 500 nM-1 μM. Using RNA-seq, we show that HCI-2509 dramatically reverses both the up- and downregulated transcriptional profiles of both EWS/FLI and EWS/ERG accompanied by the induction of apoptosis and disruption of oncogenic phenotypes modulated by EWS/FLI. We further developed a 9-gene panel based on the RNA-seq data to assess the transcriptional phenotype of HCI-2509 in additional cell lines and showed that it HCI-2509 disrupted both EWS/FLI-activated and -repressed genes similarly across the tested Ewing sarcoma cell lines. HCI-2509 impaired transformation of Ewing sarcoma cell lines in colony forming assays with IC50s from from 25 nM-1 μM. Notably, HCI-2509 displayed single-agent efficacy in multiple xenograft models. We investigated the PK/PD relationship using tumor histone H3K4 and H3K9 methylation. Taken together, these data suggest that epigenetic modulation through LSD1 inhibition may be a therapeutic strategy for Ewing sarcoma, and highlight a critical dual role for LSD1 in the oncogenic transcriptional activity of EWS/ETS proteins.

Citation Format: Emily Rose Theisen, Savita Sankar, Jared Bearss, Timothy Mulvihill, Venkataswamy Sorna, Sunil Sharma, Stephen L. Lessnick. Inhibition of LSD1 disrupts global EWS/ETS transcriptional function in Ewing sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3679. doi:10.1158/1538-7445.AM2014-3679
Abstract 3698: Hyperactive AKT pathway and reactivation of the MAPK/ERK pathway in melanoma cells resistant to dual BRAF and MEK inhibition
Authors: Wang Victoria E. , McCormick Frank , Settleman Jeffrey
Published: May 2015

In order to understand the mechanisms of resistance in melanoma cells treated with dual BRAF and MEK inhibitors, A375 and SK-Mel-28 cell lines, both harboring the BRAF V600E mutation, were treated with escalating doses of vemurafenib and cobimetinib to generate dual resistant lines. Resulting cells demonstrated 10 to 30 fold increase in resistance to the drug combination compared with the parental lines. The resistance phenotype could not be reversed by an ERK inhibitor. RNA sequencing and Western analyses revealed hyperactivation of the PI3K-AKT pathway in the A375 dual resistant cells. These cells may be re-sensitized with either a pan-PI3K inhibitor or PI3K/mTOR inhibitor. The SK-Mel-28 cells have constitutively active PI3K/AKT pathways at baseline and thus demonstrate reactivation of the MAP/ERK pathway in dual resistant cells. The therapeutic implications of these findings will be discussed.

Citation Format: Victoria E. Wang, Frank McCormick, Jeffrey Settleman. Hyperactive AKT pathway and reactivation of the MAPK/ERK pathway in melanoma cells resistant to dual BRAF and MEK inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3698. doi:10.1158/1538-7445.AM2014-3698
Abstract 370: Modeling signaling networks in tumor immunology
Authors: Clancy Trevor , Hovig Eivind
Published: May 2015

Despite decades of successful discoveries, the overwhelming molecular complexity of cells has been a significant barrier to gain a fundamental understanding of how cells work as a whole, how they are perturbed in cancer, and how they interact with signals from the immune system. To achieve a more complete understanding, will require the assistance of network modeling. By developing computational network models we can introduce cancer mutations to simulate pharmacological perturbations, or loss-of-function and gain-of-function experiments and study the dynamics of biochemical networks change under different immune signaling states. We are developing and applying rule-base (kappa) network modeling of signaling networks to study how microenvironment immune factors influence a tumor's behavior. In particular, that of immune mediated cellular senescence in tumor cells. We have built a rule-base network model of the biochemical network regulating immune mediated cell senescence in melanomas, which carry a BRAFV600 mutation in the MAPK signaling network. Using computational methods we have characterized the crosstalk with cytokine signaling networks and the crosstalk with other growth signaling networks. The development of such a computational model may help researchers understand how BRAFV600 cells in melanomas communicate with their environment by secreting various cytokines and growth factors, as it has become clear that this immune ‘secretory phenotype’ can have pro- as well as anti-tumorigenic effects.

By combining rule-based descriptions of the biochemical reactions associated to this phenotype with computer simulation we can open up new avenues for exploring such complex networks linked to melanoma progression.

Citation Format: Trevor Clancy, Eivind Hovig. Modeling signaling networks in tumor immunology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 370. doi:10.1158/1538-7445.AM2014-370
Abstract 3717: Therapeutic strategies to overcome the crizotinib resistance in ROS1-rearranged lung cancers
Authors: Katayama Ryohei , Kobayashi Yuka , Koike Sumie , Fujita Naoya
Published: May 2015

ROS1 fusion oncogene represent a novel molecular target in a small subset of non-small cell lung cancers (NSCLC).The ROS1 fusion oncogene, such as CD74-ROS1, leads to constitutive ROS1 activation with potent transforming activity. In the ongoing phase 1 clinical trial, the response rate of crizotinib, ALK/MET/ROS1 tyrosine kinase inhibitor (TKI), in the patients with NSCLC harboring ROS1 fusions was around 60%. However, despite crizotinib has shown remarkable initial responses, cancers eventually develop resistance to crizotinib. In a recent report of a clinical resistance to crizotinib, G2032R mutation in ROS1 kinase domain was identified, which is analogous to the G1202R mutation found in crizotinib-resistant ALK-rearranged lung cancers. To further identify the mechanism of resistance to crizotinib in ROS1 fusion positive NSCLC, we established a model of acquired resistance by ENU mutagenesis screening using CD74-ROS1 addicted Ba/F3 cell line models. As the results, we identified several novel crizotinib resistance mutations including G2032R mutation. To overcome the resistance, we performed the drug screening with small molecular inhibitors, and identified several inhibitors including Foretinib, cMET/VEGFR inhibitor, effectively inhibit the survival of CD74-ROS1 expressing cells. Furthermore, we also identified that some of those inhibitors can overcome the resistance caused by G2032R mutation. Thus, we have developed a comprehensive model of acquired resistance to crizotinib in ROS1 fusion harboring NSCLC and identified therapeutic strategies to overcome the resistance.

Citation Format: Ryohei Katayama, Yuka Kobayashi, Sumie Koike, Naoya Fujita. Therapeutic strategies to overcome the crizotinib resistance in ROS1-rearranged lung cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3717. doi:10.1158/1538-7445.AM2014-3717
Abstract 3720: Intrinsic and acquired resistance mechanisms of alectinib in ALK rearranged cells
Authors: Hibino Yukiko , Takaaki Sasaki , Minami Yoshinori , Janne Pasi A. , Ohsaki Yoshinobu
Published: May 2015

Background

However potency of Alectinib, a selective ALK inhibitor, for ALK rearranged NSCLC and for that with secondary mutation have been reported, intrinsic and acquired resistance are likely to emerge. Recent study showed tumor microenvironment triggered resistance to Crizotinib and TAE684. In this study, we examine whether emerging of intrinsic and acquired resistance are affected by tumor microenvironment in H3122, EML4-ALK positive lung cancer cell line, and in Alectinib-resistant clone of H3122 (AFR) which is generated in vitro. Elucidating intrinsic resistant mechanisms and preceding development of acquired resistance to understand its mechanisms will provide beneficial information for the development of novel therapeutic strategies in clinical practice.

Methods

We examined the effect of EGF produced by endothelial cells on Alectinib sensitivity of H3122 cell line, and EGF and HGF produced by fibroblasts on Alectinib sensitivity of H3122AFR by clonogenic assay. Downstream pathways of ALK in both cells were analyzed by western blotting. Combination treatments of Alectinib and other TKI were tested both in vitro and in vivo.

Results

H3122 cells which had been cultured with EGF acquired resistance to Alectinib treatment. Combination treatment of Alectinib and Erlotinib overcome EGF induced resistance and showed down-regulation in phosphorylation of AKT and ERK1/2. H3122AFR cells were also reduced their sensitivity to Alectinib at 100 uM or higher concentration when the media contained EGF or HGF. Erlotinib or Crizotinib combined with Alectinib canceled the EGF-induced or HGF-induced resistance by down-regulating phosphorylation of AKT and ERK1/2. In vivo, tumor xenografts of nude mice which had been orally administered Alectinib and Erlotinib showed tumor regression in both H3122 and H3122AFR, and those received Alectinib and Crizotinib showed regression in H3122AFR.

Conclusion

In our study, we showed ligands produced by tumor microenvironment, such as EGF and HGF, contributed the emerging of intrinsic resistance and acquired resistance by activating survival pathway. Further, the efficacy of combination treatment of Alectinib with Erlotinib or that with Crizotinib was shown. Collectively, Alectinib combined with EGFR-TKI would be an effective strategy for Alectinib-resistant lung cancer, and Alectinib with crizotinib is another considerable strategy.

Citation Format: Yukiko Hibino, Sasaki Takaaki, Yoshinori Minami, Pasi A. Janne, Yoshinobu Ohsaki. Intrinsic and acquired resistance mechanisms of alectinib in ALK rearranged cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3720. doi:10.1158/1538-7445.AM2014-3720
Abstract 3728: A discovery platform for allosteric modulators of c-Abl kinase function
Authors: Grover Prerna , Smithgall Thomas E.
Published: May 2015

The c-Abl protein-tyrosine kinase regulates intracellular signaling pathways controlling cell growth, adhesion, and responses to genotoxic stress. The kinase activity of c-Abl is repressed by intramolecular interactions involving its non-catalytic SH3 and SH2 domains. The SH3 domain interacts with a polyproline type II helix formed by the SH2-kinase linker, while the SH2 domain interacts with the C-lobe of kinase domain away from the active site. In addition, the myristoylated N-terminal cap region (Ncap) binds a hydrophobic pocket in the C-lobe of the kinase domain, clamping the SH3 and SH2 domains against the back of the kinase domain. Small molecules that allosterically regulate c-Abl kinase activity through its non-catalytic domains may represent selective probes of c-Abl function in diverse tumor types. In this study, we developed a chemical library screening assay designed to identify chemical modulators of c-Abl kinase activity that either disrupt or stabilize the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a recombinant purified protein consisting of the c-Abl Ncap, SH3 and SH2 domains plus the linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant c-Abl N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. The assay was optimized with respect to probe peptide sequence, probe and N32L protein concentrations, DMSO tolerance, and FP signal stability over time. Pilot library screens were then performed with the NCI Diversity Set III and an FDA-approved drug library (2800 compounds total). Seven compounds were identified that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds enhances c-Abl kinase activity in vitro, suggesting that it disrupts regulation of kinase activity through the non-catalytic region. These results show that screening assays based on the non-catalytic domains of c-Abl can identify small molecule regulators of kinase function.

Citation Format: Prerna Grover, Thomas E. Smithgall. A discovery platform for allosteric modulators of c-Abl kinase function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3728. doi:10.1158/1538-7445.AM2014-3728
Abstract 373: An siRNA screen identifies CHD4 as a target for epigenetic therapy
Authors: Okamoto Yasuyuki , Yamazaki Jumpei , Sato Takahiro , Cesaroni Matteo , Chung Woonbok , Garriga Judith , Jelinek Jaroslav , Katz Richard A , Issa Jean-Pierre
Published: May 2015

Background and aims: In the past decade, epigenetic mechanisms in the regulation of tumor-specific gene expression have been elucidated partially, prompting the development of therapeutic approaches in cancer cells. In this study, we used an unbiased screen to investigate therapeutic targets which might be effective combination with DNMT inhibitors in reactivating hypermethylated genes. Methods: We screened an siRNA library targeting 188 gene predicted as epigenetic regulators using colon cancer cells that harbor a GFP locus stably integrated and silenced by a hypermethylated cytomegalovirus (CMV) promoter. GFP-positive cell percentages were measured using Guava EasyCyte Plus flow analyzer software. For genome wide gene expression analysis, affymetrix microarrays were performed. DNA methylation status was evaluated by pyrosequencing analysis and Digital Restriction Enzyme Analysis of Methylation (DREAM), based on next generation sequencing analysis. To access biological effect of the combination therapy, we evaluated cell growth using several cell lines. Results: We identified the chromodomain helicase DNA-binding protein 4 (CHD4) as a target; CHD4 depletion in combination with DNMT inhibition synergistically reactivated expression of GFP and endogenously methylated genes. Synergy was most pronounced for genes with very low basal expression. DREAM analysis revealed that methylated genes with promoter CpG islands were enriched among reactivated genes compared to background genes (57% vs 27%, P<0.0001). DNA methylation levels of reactivating genes after combination therapy were identical with DNMT inhibitor monotherapy, suggesting that siCHD4 as a supplement to DNMT inhibition works by a parallel, synergistic mechanism. Combination treatment inhibited cell growth of colon cancer cell lines better than treatment with a single DNMT inhibitor with minimal effects on the cell growth of non-cancerous cell lines. Interestingly, depletion of CHD4 alone also inhibited cell growth of colon cancer cell line but not of non-cancerous cell lines. Conclusions: Combination treatment of siCHD4 and DNMT inhibitor strongly reactivated hypermethylated and silenced genes in cancer cell lines. CHD4 inhibition is a promising new approach to epigenetic targeted therapy.

Citation Format: Yasuyuki Okamoto, Jumpei Yamazaki, Takahiro Sato, Matteo Cesaroni, Woonbok Chung, Judith Garriga, Jaroslav Jelinek, Richard A Katz, Jean-Pierre Issa. An siRNA screen identifies CHD4 as a target for epigenetic therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 373. doi:10.1158/1538-7445.AM2014-373
Abstract 3732: Utilizing Bimolecular Fluorescent Complementation to Identify Inhibitors of RAL and KRAS
Authors: Ifediba Marytheresa , Burnett David , Hovda Nicholas , Burnett William , VanBrocklin Matthew
Published: May 2015

We have identified RAL activity to be vital for NSCLC survival independent of KRAS or EGFR mutation status in a large panel of adenocarcinomas and have developed a novel high throughput screening assay based on Bimolecular Fluorescent Complementation (BiFC) to identify small molecules that impair RAL function. Coupling RALA and RALB to the C-terminus of the Venus protein (VC) and coexpressing either with direct binding effector RALBP1 fused to the N-terminus of the Venus protein (VN) result in a fluorescent signal when RAL-RALBP1 binding occurs in cells. This allows for the interrogation of small molecules that disrupt RAL-RALBP1 binding interactions (resulting in loss of fluorescence) in a real time, high throughput manner. Our preliminary screen with a natural compound library identified 14 small molecules that demonstrated high activity in disrupting both RALA- and RALB-RALBP1 binding and fluorescence. We are currently investigating these compounds' mode of action and have recently expanded our assay to screen for molecules that impair mutant KRAS activity (VC-KRAS / RAF1-VN). BiFC provides a rapid, more physiologic method to identify molecules that can disrupt protein-protein interactions of targets considered ‘undruggable’, including membrane bound small GTPases such as RAL and KRAS.

Citation Format: Marytheresa Ifediba, David Burnett, Nicholas Hovda, William Burnett, Matthew VanBrocklin. Utilizing Bimolecular Fluorescent Complementation to Identify Inhibitors of RAL and KRAS. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3732. doi:10.1158/1538-7445.AM2014-3732
Abstract 3743: Bioluminescent, non-lytic, real-time cell viability assay
Authors: Duellman Sarah J. , Vidugiriene Jolanta , Zhou Wenhui , Osterman Jean , Arbit Ruslan , Bernad Laurent , Meisenheimer Poncho , Cali James J.
Published: May 2015

Cell-based assay development for cancer drug discovery continues to advance toward more innovative methods that provide efficient workflow and meaningful data. Rapid, sensitive techniques that are non-lytic and therefore facilitate multiplexing with a wide range of assays allow more data to be gained per well and conserve precious samples. Real time measurement of cell viability also allows numerous measurements over time without the need for separate plates at each time point. Real time measurements allow unique analyses including interrogation of the timing of drug killing, identification of static vs toxic drug effects, and determination of the ideal time to multiplex an additional assay. Cancer drug discovery would benefit from novel assay technologies that allow real time measurements with multiplexing capability.

We have developed a homogeneous, non-lytic, and bioluminescent method to analyze cell viability in real time through measurement of the reducing potential of the cell. The signal correlates with the number of viable cells making it well-suited for cytotoxicity studies. This Real Time Cell Viability Assay utilizes the NanoLuc luciferase enzyme, which is 100-fold brighter than either firefly (Photinus pyralis) or Renilla reniformis luciferase, and the novel substrate, furimazine. Together these reagents produce high intensity luminescence through an ATP-independent reaction.

The assay can be performed in two formats, live cell endpoint or continuous read. The live cell endpoint method is sensitive, rapid, and enables extensive multiplexing opportunities due to the non-lytic measurement and signal depletion upon cell lysis. When the assay is set up in the continuous read format, the cell viability reagents can be added at the same time as cell plating, drug dosing, or at whatever point in the assay the researcher would like to start obtaining viability readings. The luminescent signal can be continually monitored from the same wells over an extended period of time to analyze cell viability in real time.

This real time cell viability assay enables non-lytic cell viability measurements, extensive multiplexing options including both fluorescent and luminescent assays with no special filter requirements, straightforward normalization studies, conservation of precious samples and reagents, and a sensitive, real time measurement of cell viability.

Citation Format: Sarah J. Duellman, Jolanta Vidugiriene, Wenhui Zhou, Jean Osterman, Ruslan Arbit, Laurent Bernad, Poncho Meisenheimer, James J. Cali. Bioluminescent, non-lytic, real-time cell viability assay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3743. doi:10.1158/1538-7445.AM2014-3743
Abstract 3762: Downregulation of TRPC3 enhances sensitivity to cisplatin and inhibits motility in epithelial ovarian cancer
Authors: Tao Xiang , Jin Hongyan , Zhang Zhenbo , Bast Robert C. , Feng Youji , Yu Yinhua
Published: May 2015

Epithelial ovarian cancer (EOC) remains the most lethel gynecological malignancy. In the absence of an effective screening strategy, two thirds of patients are diagnosed in advanced clinical stage. Despite chemotherapy with cisplatin and paclitxel, primary or acquired chemoresistance faciltates survival of cancer cells, resulting in disease recurrence and death. Our previous studies indicate that ovarian cancer patients whose tumors overexpress transient receptor potential channel C3 (TRPC3) protein tend to relapse early and have shorter overall survival. In vitro studies indicated that follicle-stimulating hormone (FSH) induced resistance to cisplatin-induced apoptosis could be partially reversed by blocking expression of TRPC3 in EOC cell lines. To enhance sensitivity to platinum compounds, we have investigated the relationship between resistance to cisplatin and TRPC3. We have now found that expression of TRPC3 in recurrent cancer following chemotherapy is higher than in specimens from primary cytoreductive surgery. The expression of TRPC3 in the cisplatin-resistant EOC line A2780cp is higher than in the sensitive parental cell line A2780. An in vitro study showed that transient treatment of the EOC cell line HEY with cisplatin upregulated TRPC3 mRNA and membrane localized protein. Induced cisplatin-resistant cell line HEYcp augmented the expression of TRPC3 and enhanced invasive capacity in cell migration assays. Knockdown of TRPC3 expression in EOC cell lines with specific siRNA or miRNA decreased cell migratory ability and enhanced cisplatin-induced apoptosis when compared to EOC cells treated with scrambled control RNA. Our data indicate that TRPC3 may play an important role in cisplatin-resistance of EOC and could serve as a therapeutic target to resensitize cancer cells to cisplatin in culture and in xenografts.

Citation Format: Xiang Tao, Hongyan Jin, Zhenbo Zhang, Robert C. Bast, Youji Feng, Yinhua Yu. Downregulation of TRPC3 enhances sensitivity to cisplatin and inhibits motility in epithelial ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3762. doi:10.1158/1538-7445.AM2014-3762
Abstract 3795: Cabozantinib affects multiple signaling pathways in glioblastoma and is effective in a subset of xenograft tumors
Authors: deCarvalho Ana C. , Arnold Kimberly , Mueller Claudius , Petricoin Emanuel F. , Poisson Laila M. , Mikkelsen Tom
Published: May 2015

Background: Glioblastoma (GBM) is the most common and aggressive form of brain tumor characterized by poor prognosis and high recurrence rate. Several new targeted agents are being considered for the adjuvant treatment of GBMs. Cabozantinib, a multi-kinase currently in clinical trials for several malignancies, including glioblastoma, targets c-Met and VEGFR2 receptors, as well as Ret, Kit, Flt-1/3/4, Tie2, and AXL. Our goal is to first evaluate sensitivity and molecular response of patient-derived GBM cancer stem cells (CSCs) to XL184 in vitro. Anti-tumor efficacy of cabozantinib as a single agent and in combination with the DNA-alkylating agent temozolomide (TMZ) in orthotopic xenografts was then evaluated.

Methods: Neurospheres enriched in CSCs were cultured from resected GBM tumors. Sensitivity to cabozantinib was determined in vitro. Cells were treated (IC40) in triplicate, and cell lysates were analyzed by reverse phase protein microarrays. GBM CSCs were implanted intracranially into nude mice. Cabozantinib was administered by oral gavage at a dose of 60mg/kg for 4 weeks (5 days/week) as a single agent or in combination with 40mg/kg TMZ. Tumor growth and response to treatment were monitored by non-invasive in vivo biolumiescent imaging (BLI) using the Xenogen IVIS System (Caliper Life Sciences), and overall survival.

Results: Sensitivity to cabozantinib treatment varied for the different GBM CSCs. From 70 proteins and phosphoproteins measured, 29 distributed among several signaling pathways were significantly altered after treatment in both resistant and sensitive GBM CSCs. Cabozantinib single agent treatment reduced GBM tumor growth and increased mouse survival in two xenograft lines. Cabozantinib monotherapy reduced tumor size, as measured by BLI, but had no significant effect on overall survival for another xenograft line, however, the combination treatment resulted in sensitization of these xenografts to TMZ treatment.

Conclusion: The use of multi-kinase inhibitors is a promising strategy for GBMs, and given the range of responses, identifying predictive biomarkers, though challenging, will be invaluable for patient stratification.

Citation Format: Ana C. deCarvalho, Kimberly Arnold, Claudius Mueller, Emanuel F. Petricoin, Laila M. Poisson, Tom Mikkelsen. Cabozantinib affects multiple signaling pathways in glioblastoma and is effective in a subset of xenograft tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3795. doi:10.1158/1538-7445.AM2014-3795
Abstract 3799: Urokinase kringle-derived peptide UP-7 potently inhibits angiogenesis and tumor growth
Authors: Kim Hyun-Kyung , Naidansuren Purevjargal , Lee Seung Woo , Joe Young Ae
Published: May 2015

The recombinant kringle domain of urokinase-type plasminogen activator (UK1) has been shown to inhibit angiogenesis in vitro and in vivo and suppress brain tumor in vivo. UK1 consists of 83 amino acids and has 2 short helices, rudimentary ß-sheets and one extended antiparallel ß-sheets regions. In order to identify functional core sequence of the UK1 for angiogenesis inhibition, we examined the anti-angiogenic and anti-tumor activities for the seven UK1-derived peptides. Among the tested peptides, UP-7 potently inhibited the proliferation and migration of endothelial cells in vitro and suppressed vessel formation in Matrigel plugs in vivo. However, such anti-angiogenic activities were not exerted by the scrambled peptide UP7-SC7 or UP7-derived shorter peptides. UP-7 also suppressed VEGF-induced phosphorylation of FAK and ERK1/2 in endothelial cells. In a NCI-H460 lung cancer xenografted animal model, UP-7 effectively inhibited tumor growth. In addition, treatment of mice bearing MDA-MB231 tumor with UP-7 peptide resulted in a dose-dependent inhibition of tumor growth. Taken together, these results suggest that UK1- derived novel peptide UP-7 can be used for treatment of cancers. (NRF-2012R1A1A2007175).

Citation Format: Hyun-Kyung Kim, Purevjargal Naidansuren, Seung Woo Lee, Young Ae Joe. Urokinase kringle-derived peptide UP-7 potently inhibits angiogenesis and tumor growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3799. doi:10.1158/1538-7445.AM2014-3799
Abstract 3800: Antiproliferative effect of fingolimod (FTY720) in human prostate cancer cells: Insights to estrogen metabolism in situ
Authors: Allam Rasha M. , Mosli Hisham A. , Khalifa Amany E. , Nofal Salwa M. , Sharaf Ola A. , Abdel-Naim Ashraf B. , Al-Abd Ahmed M.
Published: May 2015

Sphingokinase-1/sphingosine-1-phosphate pathway contributes significantly to progression of different types of cancer which has been recently reported to influence estrogen level in breast cancer cells. Current evidence suggests that estrogen (E2) and its metabolites, especially catechol metabolites, are playing a key role in the proliferation and carcinogenesis of the prostate glands. FTY720 is Sphingokinase-1inhibitorwhich possess anticancer properties in various tumor cell types. The aim of this study is to investigate the chemotherapeutic effect of FTYY720 against prostate cancer cells with emphasis on estrogenic microenvironment in-situ. The IC50 of FTY720 in DU145 cell line was4.2±1.4µg/ml. The concentration of estrogen and its metabolites were assayed in the media over DU145 cells using liquid chromatography tandem mass (LC/MS) technique. FTY720 treatment resulted in a dramatic decrease in estradiol (E2) and estrone (E1) levels compared to control untreated cells (2652.76±115.62 vs. 725.69±1.28 and 283.36±4.18vs. 150.32±8.96ng/g, respectively); however, it had no significant effect on the concentration of Estriol(E3). In terms of catechol estrogens, 4-hydroxyEstradiol (4-OHE2) and 2-hydroxy Estradiol (2-OHE2) formation was significantly decreased after treatment withFTY720 compared to control cells (25.42±2.40 vs. 7.45±0.82 and 485.93±3.10 vs. 477.42±4.24 ng/g), respectively. FTY720 had no significant effect on formation of 16α-hydroxyEstrone (16 α-OHE1). On the other hand, treatment with FTY720 significantly increased the concentration of 2-methoxyestrone (2-MOHE1) and 2-methoxyestradiol (2-MOHE2) compared to control cells (5.45±0.12 vs. 29.94±2.97 and165.62±5.67 vs. 269.97±15.85), respectively..The expression of catechol estrogen synthesizing enzymes (CYP19, CYP1A1 and CYP1B1) was significantly decreased within prostate cancer cells after treatment with FTY720 compared to untreated counterpart. On the other hand, the expression level of catechol estrogen degrading enzyme (COMT) was significantly decreased. Cell cycle analysis of DU145 cells showed arrest at G1 phase with significant decrease in the G2-M population due to treatment with FTY720. In conclusion, FTY720 induced anti proliferative effect in prostate cancer cells might be attributed partly to its influence on estrogen metabolism in-situ.

Citation Format: Rasha M. Allam, Hisham A. Mosli, Amany E. Khalifa, Salwa M. Nofal, Ola A. Sharaf, Ashraf B. Abdel-Naim, Ahmed M. Al-Abd. Antiproliferative effect of fingolimod (FTY720) in human prostate cancer cells: Insights to estrogen metabolism in situ. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3800. doi:10.1158/1538-7445.AM2014-3800
Abstract 3814: FAK inhibition targets nucleostemin, a nucleolar protein, impacting breast cancer spheroid growth and tumor progression
Authors: Tancioni Isabelle , Uryu Sean , Miller Nichol L. G. , Lawson Christine , Jean Christine , Chen Xiao Lei , Schlaepfer David D.
Published: May 2015

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that controls cell proliferation and survival downstream of integrin-matrix receptors. However, under deprivation of anchorage, FAK translocates to the nucleus. The nucleolus, a non-membrane structure within the nucleus, is important in the control of ribosome biogenesis, regulates the sequestration of nuclear proteins, and increased nucleolar size is a marker of aggressive tumors. Nucleostemin, a nucleolar-localized protein, acts to modulate cell cycle progression and anchorage-independent cell growth. Although many drugs have low efficacy on tumor cells cultured in suspension, nanomolar small molecule FAK inhibitor (PF-271 or PND-1186) addition can prevent anchorage-independent growth of tumor cells as spheroids through mechanism(s) that remain unresolved. Here, we show that pharmacological FAK inhibitor (FAK-I) treatment or kinase-dead (KD) FAK re-expression analyses reduced breast cancer (MDA-MB-231 and 4T1-L) anchorage-independent cell growth with a G0/G1 cell cycle block. This was associated with a specific decrease in nucleostemin protein levels. As FAK-KD forms a complex with nucleostemin, and proteasome inhibitor treatment increased nucleostemin levels in FAK-KD cells, FAK-KD may function as a scaffold to facilitate nucleostemin degradation. In orthotopic breast cancer mouse tumor models, oral FAK-I administration or FAK-KD expression resulted in smaller tumors, decreased Ki67 positive cells, and lower levels of tumor-associated nucleostemin. Ongoing experiments are focused on understanding the molecular mechanisms of FAK nucleolar localization and the regulation of nucleostemin protein levels. Our results point to a novel function of FAK in nucleolar function, spheroid growth, and breast cancer progression.

Supported by NIH CA102310 and Susan G. Komen for the Cure (KG111237).

Citation Format: Isabelle Tancioni, Sean Uryu, Nichol L. G. Miller, Christine Lawson, Christine Jean, Xiao Lei Chen, David D. Schlaepfer. FAK inhibition targets nucleostemin, a nucleolar protein, impacting breast cancer spheroid growth and tumor progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3814. doi:10.1158/1538-7445.AM2014-3814
Abstract 3818: Tumor budding and E-cadherin expression are useful predictors of nodal involvement in T1 esophageal squamous cell carcinoma
Authors: Kubo Naoshi , Ohira Masaichi , Sakurai Katsunobu , Toyokawa Takahiro , Tanaka Hiroaki , Muguruma Kazuya , Yashiro Masakazu , Maeda Kiyoshi , Onoda Naoyoshi , Hirakawa Kosei
Published: May 2015

Background The endoscopic treatment has been increasingly used for T1 esophageal squamous cell carcinoma (ESCC). However, this therapy is sometimes incomplete if the depth of the T1 primary tumor reaches the muscularis mucosae (MM) or submucosal layer (SM) because these tumors have relatively high incidences of lymph node metastasis. However, no previous report on the predictive factor of nodal metastasis determined by evaluating primary tumor specimens of patients with ESCC is available.

Patients and Methods A total of 55 patients with T1 ESCC invading as deep as the submucosal layer who underwent curative esophagectomy were examined. We investigated the significance of the immunohistochemical stainings of VEGF-C and E-cadherin in the primary tumor and tumor budding for prediction of nodal metastasis.

Results Metastasis to the regional lymph nodes was observed in 26 cases (47.3%) in this setting. VEGF-C expression and reduced E-cadherin expression in the primary tumor was observed in 32 (58.1%) and 38 cases (69.1%), respectively. High-grade tumor budding was observed in 29 cases (52.7%). E-cadherin expression and tumor budding were closely correlated with nodal metastasis (p = 0.039 and <0.01 respectively), whereas VEGF-C expression tended to correlate with lymph node metastasis (p = 0.055). In addition, high-grade tumor budding was significantly correlated (p < 0.01) with reduced E-cadherin expression. The accuracies of tumor budding and E-cadherin expression for nodal metastasis were 67.3% and 65.4% respectively, which were comparable with those of lymphatic involvement (63%). Tumor budding (p < 0.01), but not E-cadherin and VEGF-C expression, was significantly correlated with poor survival.

Conclusion After the endoscopic treatment, additional therapy, such as surgery or chemoradiotherapy, may be required if reduced E-cadherin expression and high-grade tumor budding are observed in the primary tumor specimen.

Citation Format: Naoshi Kubo, Masaichi Ohira, Katsunobu Sakurai, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Masakazu Yashiro, Kiyoshi Maeda, Naoyoshi Onoda, Kosei Hirakawa. Tumor budding and E-cadherin expression are useful predictors of nodal involvement in T1 esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3818. doi:10.1158/1538-7445.AM2014-3818
Abstract 3837: Innovative rapid gene methylation analysis of surgical margin tissues in head and neck cancer
Authors: Hayashi Masamichi , Guerrero-Preston Rafael , Kahn Zubair , Li Xiufeng , Ahn Julie , Goldsmith Marla , Michailidi Christina , Koch Wayne
Published: May 2015

Background: To secure negative surgical margins is the primary goal of surgical cancer treatment. However, tumor recurrence sometimes occurs even with histologically negative surgical margins. In order to detect submicroscopic residual cancerous cells intra-operatively, a time saving molecular biological approach is required.

Methods: We established an innovative rapid Quantitative methylation PCR (QMSP) assay, which consists of remarkably time-minimized DNA extraction, bisulfite treatment and QMSP quantification. In order to assess the feasibility of completing the rapid assay during a surgical procedure, 10 serial specimens of primary head and neck cancers were evaluated by both rapid QMSP and conventional QMSP.

Results: The DNA product quality of the most time saving procedure was as good as that of a conventional procedure. In the QMSP assay, target gene methylation and reference gene methylation were equally detected by both rapid and conventional methods. Finally, two frequently methylated genes in head and neck cancers, homeobox A9 (HOXA9) and endothelin receptor type B (EDNRB) were analyzed in 10 head and neck tumors, margin tissues and normal tissues. Relative QMSP values of rapid and conventional QMSP were quite similar in almost all tumors, margins and normal tissues. The average total time for the rapid QMSP procedure was less than 3 hours. Although postoperative follow-up periods are less than a year, local tumor recurrence occurred in one case whose margin tissue was QMSP positive.

Conclusion: This innovative rapid QMSP yields acceptable levels of methylation detection and can be completed during the operative window. The procedure promises to become a useful clinical tool, if the methylated surgical margins are revealed to correlate with the local tumor recurrence in a larger cohort.

Citation Format: Masamichi Hayashi, Rafael Guerrero-Preston, Zubair Kahn, Xiufeng Li, Julie Ahn, Marla Goldsmith, Christina Michailidi, Wayne Koch. Innovative rapid gene methylation analysis of surgical margin tissues in head and neck cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3837. doi:10.1158/1538-7445.AM2014-3837
Abstract 3839: Oxidative damage in predicting early adverse skin reactions from adjuvant radiotherapy in a tri-racial/ethnic breast cancer population
Authors: Nelson Omar L. , Takita Cristiane , Wright Jean , Lee Eunkyung , Hu Jennifer J.
Published: May 2015

Adjuvant radiotherapy (RT) following breast conservation surgery has significantly improved survival. However, may patients develop adverse skin reactions, pain, breast edema and poor cosmetic results that impact quality of life. Inter-individual variability in the development of RT-induced adverse reactions in normal tissue is well-documented for both acute and late effects. Therefore, we evaluated the first 161 patients in an ongoing prospective study of conservatively managed breast cancer patients using the NCI CTCAE v3.0 criteria. We recorded patient characteristics, smoking history, body mass index (BMI), and treatments. We pilot tested urinary 8-hydroxydeoxyguanosine (8-OHdG) as an oxidative damage marker in predicting RT-induced skin toxicity. Women younger than 50 years of age had a significantly (p=0.016) higher post-RT skin toxicity (grade 2); higher proportion of African Americans have grade 3 skin (p=0.008) compared to other races; and obesity (BMI>30) was associated with worse skin toxicity. In multivariate analysis, women with a higher pre-RT 8-OHdG had a significantly higher risk of worse skin toxicity at week 3 (OR=11.77; 95C%CI=1.21-114.45; p=0.034). Obesity (BMI>30) was associated with worse skin toxicity at the end of RT (OR=2.27; 95C%CI=1.14-4.50; p=0.019). This is the first study evaluating oxidative damage in RT-induced skin toxicity. We reported significant racial/ethnic differences in urinary 8-OHdG levels and a significant association between pre-RT 8-OHdG and worse skin toxicity at week 3. If confirmed in our larger ongoing study of 1,255 patients, our study will lead to effective treatment strategies and improve quality of life of breast cancer patients undergoing RT.

Citation Format: Omar L. Nelson, Cristiane Takita, Jean Wright, Eunkyung Lee, Jennifer J. Hu. Oxidative damage in predicting early adverse skin reactions from adjuvant radiotherapy in a tri-racial/ethnic breast cancer population. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3839. doi:10.1158/1538-7445.AM2014-3839
Abstract 3844: Venous thromboembolism in patients with pancreatic cancer
Authors: Yoon Seug Yun , Lee Namsu , Kim Sook-Ja , Cheong Hee-Jeong , Kim Kyoung Ha , Won Jong-Ho , Park Hee Sook
Published: May 2015

Background:

Venous thromboembolism (VTE) is a critical complication of malignant disease. Pancreatic cancer is one of the cancers most commonly associated VTE. In general, the VTE incidence rate of Asians is lower than that of Caucasians. In 2007, a Korean study reported that only four cases of VTE (5.3%) occurred in Seventy five patients with advanced pancreatic adenocarcinoma. We evaluated VTE incidence in pancreatic cancer and characteristics of pancreatic cancer patients with VTE.

Method:

We retrospectively reviewed the medical records of patients with histopathologically proven pancreatic cancer from January 2006 to December 2012 at Soonchunhyang university hospital. We detected VTE through CT (chest CT, pulmonary embolism CT) and low extremity ultrasoundgraphy.

Results

Five hundred and fourteen patients with pancreatic adenocarcinoma were enrolled. (M: F, 300:214, localized: locally advanced: metastatic=31:230:253, mean age: 66.7 years). Ninety six of 514 patients (18.6%, symptomatic: aymptomatic=38:58, PE: DVT: PE+DVT: visceral thrombosis=20:19:19:38) were diagnosed as VTE. At the time of DVT diagnosis, cancer status of 50 patients cancer was progression, and that of 15 was stable. Thirty one patients were diagnosed with the pancreatic cancer and VTE, at the same time. They all had metastatic lesions. Fifty VTE patients were treated with antithrombotic therapy. Ninety three of 96 patients died, and three of them have probability that cause of death was VTE. The others died of pancreatic cancer progression. From pancreatic cancer diagnosis to VTE diagnosis, the period is 1.7month. (95%CI 1.1-2.3 month). Median overall survival (OS) was not significantly different between pancreatic cancer with VTE or without VTE. OS was significantly longer VTE patients after pancreatic cancer diagnosis than VTE patients with pancreatic cancer at the same time (10.73m vs. 1.7, p=0.00).

Conclusion

The incidence of VTE (18.6%) in Soonchunhyang university hospital with pancreatic cancer was not lower than that in western groups.

Citation Format: Seug Yun Yoon, Namsu Lee, Sook-Ja Kim, Hee-Jeong Cheong, Kyoung Ha Kim, Jong-Ho Won, Hee Sook Park. Venous thromboembolism in patients with pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3844. doi:10.1158/1538-7445.AM2014-3844