bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-316: Pharmacokinetic modeling optimizes direct inhibition of the ‘undruggable’ target EWS-FLI1 of Ewing Sarcoma
Authors: Hong S. Peter , Gamble S. Ellen , Hong Sung-Hyeok , Kobs Christina L. , Elsass Karen E. , South Natalie L. , Monroe Philip J. , Üren Aykut , Toretsky Jeffrey A.
Published: August 2012

Chimeric transcription factors have long been considered ideal anti-cancer targets since they are only present in tumor cells, however, their lack of enzymatic activity has placed them in a category of ‘undruggable’ proteins. The EWS-FLI1 fusion protein of Ewing Sarcoma (ES) has been validated as an anticancer target both alone and as a partner of RNA Helicase A (RHA). Our prior work identified the small molecule YK-4-279 as a specific inhibitor of EWS-FLI1 by blocking the interaction with RHA. Blocking this interaction leads to apoptosis. Data suggests that relatively long exposures of drug are necessary to keep EWS-FLI1 and RHA apart. We have modeled YK-4-279 treatment using cell lines (TC71, TC32, A4573, SK-ES, and RD-ES) to establish the duration of YK-4-279 exposure that leads to apoptosis. Apoptosis was measured by caspase-3 cleavage. A validated plasma detection method allows for HPLC measurement of YK-4-279. Pharmacokinetic (PK) models of YK-4-279 for both intraperitoneal (IP) and intravenous (IV) administration were developed in SD rats and BL6 mice. ES cell lines were exposed to YK-4-279 over a time-course, followed by a caspase-3 activity assay that demonstrated a minimum of 16 hours exposure was necessary to achieve maximal apoptosis using either 3 or 10 microM compound. A dose-dependency assay demonstrated that while apoptosis could be achieved with 30 - 80 microM YK-4-279 after 4 hours of treatment, caspase-3 activity was less than or equal to 3 microM for 16 hours. Rat PK modeling demonstrated a t1/2 of 30 minutes following IV administration. BL6 mice demonstrated similar kinetics to the rat. SCID/bg mice, which are necessary for tumor efficacy studies, demonstrated approximately 50% faster clearance than either rat or BL6 mice. Absolute bioavailability for IP administration was 41%. Models that use cell culture based targets for plasma levels and duration of exposure will be created to optimize IP dosing regimen. The optimized IP dosage and dosing intervals will be evaluated in tumor bearing animals in order to advance development of YK-4-279. The results of these studies will be used to guide toxicology studies in animals. In addition, pharmacodynamics models are being developed to compare YK-4-279 levels with functional activity. The combined results of these investigations will lead to human clinical trials for this first-in-chemical class, first-in-mechanism drug candidate.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-316. doi:1538-7445.AM2012-LB-316
Abstract LB-353: Development of thymidylate synthase ternary complex-specific antibody: A new way to tailor 5-fluorouracil dosing
Authors: Patel Kalpesh , Yerram Sashidhar R. , Kern Scott E.
Published: August 2012

Introduction: 5-Fluorouracil (5FU) is a fluorinated nucleoside analogue used in cancer treatment. 5FU metabolites modify thymidylate synthase (TS) to form a ternary complex and inhibit its activity. Despite its benefits, major side effects are common and can lead to discontinuation of 5FU therapy. Although dose, schedule, and mode of application of 5FU can be adjusted to minimize toxicity, there has been a little done to tailor 5FU dosage for cancer therapy. Aims: To develop a ternary complex-specific monoclonal antibody. To develop a test quantifying drug-complexed TS level so as to measure the action of 5FU at the cellular level. Methods: Recombinant TS was produced in E. coli. In-vitro, the ternary complex was created. A specific rat monoclonal antibody was raised against drug-complexed TS. ELISA and immunoblot (IB) assays were validated using 5FU-treated cells. Tumors were developed in nude mice by injection of RKO and MiaPaCa2 cells. We treated tumors in mice with 5FU at different doses and assayed the tumor upon harvest. Results: The drug-complexed TS migrates slower than native TS in protein gels. Our novel ternary complex-specific rat monoclonal antibody (10C4) specifically recognized drug-complexed TS in ELISA and IB in both cultured cells and tumors treated with 5FU. Commercially available TS-specific antibody recognized both forms of TS. Conclusion: We developed and characterized the first thymidylate synthase ternary complex-specific rat monoclonal antibody. Implications: Quantification of drug-complexed TS may be possible with use of the new antibody. The actual action of 5FU at the cellular level can now be determined readily. It may become possible to calculate individualized 5FU doses in the clinic using advances in the technology. Such developments might decrease the incidence of toxicity and increase drug effectiveness during 5FU treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-353. doi:1538-7445.AM2012-LB-353
Abstract LB-370: Genetically engineered oncolytic Edmonston strain of measles strain harboring the wild-type N, P, L genes (MV-NPL) effectively target lung cancer stem cells
Authors: Inoue Hiroyuki , Hijikata Yasuki , Yasunari Keisuke , Sakamoto Akira , Miyamoto Shohei , Nosaki Kaname , Matsumura Yumiko , Takayama Koichi , Nakanishi Yoichi , Tani Kenzaburo
Published: August 2012

Increasing evidence suggests that only a small percentage of cancer cells in solid tumors have tumorigenic potential. These highly tumorigenic cells share with normal stem cells the ability to proliferate and give rise to diversified tumor cell types including those with the capacity for self-renewal. These cells are termed cancer stem cells (CSCs) and are believed to contribute to CSCs resistance to conventional therapies. Therefore, specific targeting of CSCs is an attractive and novel therapeutic approach. Measles virus Edmonston strain (MV-Edm) is thought to possess remarkable oncolytic activity that selectively destroys human tumor cells. Recently, we succeeded and reported the development of novel oncolytic measles virus (MV) by genetic engineering of MV-Edmonston strain (MV-Edm), that have demonstrated its remarkable oncolytic activity against wide ranges of human tumor cells, with the N, P, and L genes of wild-type MV (MV-NPL). The oncolytic activities of the MV-NPL on human renal cell carcinoma were superior to those of MV-Edm both in vitro and in vivo studies even in the presence of IFN[[Unsupported Character - ‐]]α[[Unsupported Character - ]](Xing, M. et. al. Mol Ther 2010). Here, we demonstrated that lung CSCs, side population fraction of human lung cancer cells of A549 (A549-SP), expressed high level of CD46 required for MV-NPL infection and MV-NPL had a superior oncolytic capacity against both A549-SP and A549-NSP (non SP) cells in a time and dose-dependent manner without damaging normal lung cells. Our results of pan-caspase inhibiton assays showed the partial contribution of caspases-dependent apoptosis in the oncolysis of A549-SP cells by MV-NPL, even though the expression levels of anti-apoptotic protein of both XIAP and Mcl-1 were higher in A549-SP cells than A549-NSP cells. Additionally, we demonstrated that an addition of either PI3K inhibitor or MEK inhibitor further enhanced the oncolytic effects of MV-NPL. Furthermore, our in vivo studies using nude mice bearing A549 xenografts revealed that serial intratumoral MV-NPL administrations exhibited significant antitumor effects without severe side effects. Collectively, these data suggested that genetically engineered MV-NPL could be a promising oncolytic agent to effectively eradicate lung CSCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-370. doi:1538-7445.AM2012-LB-370
Abstract LB-373: Parp1 dependent regulation of chromatin remodeling by the ALC1 (Amplified in Liver Cancer) oncogene
Authors: Gottschalk Aaron J. , Trivedi Rushi D. , Conaway Joan W. , Conaway Ronald C.
Published: August 2012

The human ALC1 (Amplified in Liver Cancer 1) gene was originally identified as a gene present on a human chromosome 1q21 region amplified in a large fraction of hepatocellular carcinomas. Subsequent studies found that overexpression of the ALC1 protein was found to be oncogenic in human cells and mice, although the mechanism of action of ALC1 in oncogenesis is not known. The human ALC1 gene encodes a member of the SNF2 superfamily of ATPases. The ALC1 protein is unique among SNF2 family members because it includes a macrodomain that is capable of binding selectively to poly(ADP-ribose) (PAR). We and others have also previously demonstrated that ALC1 possesses DNA-dependent ATPase and ATP-dependent nucleosome remodeling activities that are strongly dependent on the presence of poly(ADP-ribose) polymerase PARP1 (or the closely related PARP2) and NAD+ within in vitro assays. Our preliminary investigation of the mechanism of PARP1- and NAD+-dependent activation of ALC1 DNA-dependent ATPase and nucleosome remodeling activities revealed (i) that both the ALC1 SNF2 ATPase domain and an intact macrodomain capable of binding PAR are essential for activation by PARP1 and NAD+, (ii) that activation of ALC1 in the presence of PARP1 and NAD+ results in increased affinity of ALC1 for DNA and nucleosomes, and (iii) that autoPARylated PARP1 is most likely the source of PAR required for activation of ALC1. These preliminary observations provided some insight into basic features of the function of the ALC1 protein, but they left unresolved major questions concerning the mechanism by which ALC1 is activated by PARP1 and NAD+ to remodel nucleosomes. In our recent studies, we extend our investigation of the mechanism by which ALC1 is “activated” to remodel nucleosomes by PARP1 and NAD+. Our findings argue that (i) ALC1 activation requires assembly of a stable ALC1.autoPARylated PARP1.nucleosome intermediate, (ii) that ALC1 regulates association of autoPARylated PARP1 with this intermediate, and (iii) stable binding of the ALC1 macrodomain to autoPARylated PARP1 is critical for ALC1 activation. Taken together, our findings are consistent with the model that a stable ALC1.autoPARylated PARP1 heterodimer is the active species in ALC1-dependent ATPase and nucleosome remodeling. These findings provide further important insight into the mechanism of action of this poorly understood oncogene.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-373. doi:1538-7445.AM2012-LB-373
Abstract LB-380: Distinct methylation pattern in early-onset colorectal cancer patients without detectable germline mutation
Authors: Chan Tsun-Leung , Li Carmen , Yuen Siu-Tsan , Chan Anthony KW , Tsui Wendy WY , Chan Annie SY , Leung Suet-Yi
Published: August 2012

Colorectal cancer (CRC) is currently the second most common cancer and mortality rate of all cancers in Hong Kong. We have previously reported a higher incidence of early-onset colorectal cancer patients compare with other ethnic groups, which may be caused by genetic or epigenetic predisposition in the Chinese population. Despite we confirmed Lynch Syndrome (LS) plays a significant role in it, pathogenic event of roughly 20% of young patients with MSI tumour cannot be identified. These patients are still susceptible to novel genetic or epigenetic predisposition of CRC. In this study, we compared the epigenetic signature of CRC patients below and above the age of 50 years old, who developed High level of microsatellite instability (MSI-H) and loss of MLH1 expression. The CpG island methylator phenotype (CIMP), regional methylation events, Braf and Kras mutation status have been examined. Among 12 cases of the early-onset patients, unexpectedly 5 of them showed constitutive MLH1 promoter hypermethylation versus 1 in 26 cases of the late-onset control patients by pyrosequencing (p-value < 0.01). CIMP-High status, which has been frequently reported in late-onset patients, was found to be 25% and 77% in the early and late-onset groups respectively (p value < 0.001). Strikingly, regional methylation coincides with the CIMP-High cases and MLH1 promoter methylation was the unique epimutation event in the chromosomal region encompass MLH1 in most of the early-onset patients. We are the first to demonstrate that CIMP status and MLH1 regional methylation are a distinct phenotype in the patient with early-onset CRC irrespective of the constitutive status of MLH1 promoter methylation event. Our data suggest that the tumorigenesis of early-onset CRC is significantly different from patients with late-onset CRC despite they are all classified as MSI-H and loss of expression of MLH1. Lastly, epigenetic signature of most of the early-onset CRC patients is indistinguishable from the patient with constitutive MLH1 promoter methylation. It is unclear epigenetic predisposition contribute to the high incidence of early-onset CRC. Further investigation for additional markers should be performed as routine Kras and Braf mutation status were uninformative.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-380. doi:1538-7445.AM2012-LB-380
Abstract LB-389: The function of histone demethylase RBP2 in breast cancer metastasis and drug resistance
Authors: Cao Jian , Liu Zongzhi , Zhao Minghui , Nguyen Don , Yan Qin
Published: August 2012

Metastasis and drug resistance are two major challenges in cancer therapies. The occurrence of these two events requires cancer cells to gain new mutations or change their expression profiling, or both, adapt to new environment (i.e. distal organs or drug treatments). These changes likely follow Darwin's evolutionary theory: heterogeneous of cancer cells are generated randomly and more aggressive cells are selected by the tissue environment. Stable inheritance of genome and epigenetic states could delay metastasis and drug resistance. Trimethylated lysine 4 in histone H3 (H3K4me3) is the epigenetic marker for transcriptionally active promoters and the binding site for transcriptional activators. Removing the methyl groups from trimethylated H3K4 on gene promoter regions is a common epigenetic strategy for gene silencing. The JARID1 family members are to date the only known proteins responsible for catalyzing demethylation of trimethylated H3K4. Therefore they are essential in cell differentiation and development. Even though JARID1A (RBP2) was implicated in tumorigenesis of certain cancer types, its exact roles are still poorly understood. I hypothesized that RBP2 contributes to changes of epigenetic states during the occurrence of metastasis and drug resistance. Consistent with my hypothesis, I found that RBP2 is highly expressed in metastatic breast cancer cells and Herceptin tolerant population. Loss of RBP2 decreased lung metastatic gene signature and increase drug sensitivity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-389. doi:1538-7445.AM2012-LB-389
Abstract LB-4: Preclinical development of spherical nucleic acids, a novel RNAi-based nanomedicinal platform, as anti-glioma therapeutics
Authors: Stegh Alexander H.
Published: August 2012

Patients with Glioblastoma (GBM) survive for a mere 14-16 months. Conventional and targeted therapies, foremost inhibitors of receptor tyrosine kinases (RTK-Is) have been ineffective in glioma clinical trials, contributing to a near 100% failure rate of GBM drug development. Impediments to clinical successes are the lack of systematic identification of genes with critical roles in therapy (apoptosis) resistance, and the dearth of innovative technologies to deliver therapeutics to glioma elements, and to effectively target important apoptosis regulators in established gliomas. Here, we describe the development and pre-clinical characterization of a novel RNAi-based nanomedicinal platform (termed spherical nucleic acids, SNAs) to target aberrantly expressed oncogenes in GBM. SNAs consist of polyvalent gold nanoparticles with diameters of ∼13 nm, which are densely functionalized with RNAi molecules via oligoethylene glycol-thiol linkers. Our detailed physico-chemical and cell biological characterization revealed that SNAs exhibit highly efficient cellular uptake and robust serum stability without the use of auxiliary transfection strategies or chemical modifications, less induction of innate immune responses, reduced off-target effects, and minimal systemic toxicity in rodents. In proof-of-priciciple studies, we utilized SNAs to neutralize the expression of anti-apoptotic Bcl-2 proteins, i.e., canonical family members, such as Bcl-2, Bcl-xL, and the atypical member Bcl2L12. We show highly efficient uptake of SNAs into various primary and transformed glial cell lineages in vitro, including patient- and glioma mouse model-derived tumor neurospheres (TNS). Importantly, SNAs provoked robust and persistent knockdown of Bcl-2 family members on mRNA and protein levels, and robustly sensitized glioma cells toward therapy-induced apoptosis. Finally, Magnetic Resonance Imaging and Inductively Coupled Plasma Mass Spectrometry of SNAs co-functionalized with gadolinium revealed that intravenously administered SNAs penetrated the blood-brain- and blood-tumor-barriers, and robustly disseminated xenogeneic glioma explants. High-level accumulation of Bcl2L12-targeting SNAs within glioma elements was associated with increased intratumoral apoptosis, and consequently resulted in reduced glioma formation and prolonged survival of xenografted mice. Thus, silencing of anti-apoptotic signaling driven by members of the Bcl-2 family using nano-RNAi represents a novel therapeutic approach to overcome therapy resistance and to halt progression of GBM.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-4. doi:1538-7445.AM2012-LB-4
Abstract LB-400: The effects of hepatitis B virus integration into the genomes of hepatocellular carcinoma patients
Authors: Zhang Zemin
Published: August 2012

Hepatitis B virus (HBV) infection is a leading risk factor for hepatocellular carcinoma (HCC). HBV integration into the host genome has been reported but its scale, impact and contribution to HCC development is not clear. Here, we sequenced the tumor and nontumor genomes (>80X coverage) and transcriptomes of four HCC patients and identified 255 HBV integration sites. Increased sequencing to 240X coverage revealed a proportionally higher number of integration sites. Clonal expansion of HBV-integrated hepatocytes was found specifically in tumor samples. We observe a diverse collection of genomic perturbations near viral integration sites, including direct gene disruption, viral promoter-driven human transcription, viral-human transcript fusion and DNA copy number alteration. Thus, we report the most comprehensive characterization of HBV integration in hepatocellular carcinoma patients. Such widespread random viral integration will likely increase carcinogenic opportunities in HBV-infected individuals.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-400. doi:1538-7445.AM2012-LB-400
Abstract LB-402: New fusion genes in lung adenocarcinoma revealed from next generation RNA-seq
Authors: Ju Young Seok , Lee Won-Chul , Shin Jong-Yeon , Kim Jong-Il , Kang Jin-Hyoung , Kim Young Tae , Seo Jeong-Sun
Published: August 2012

Cancer is a genomic disease. A subset of somatic driver mutations has been reported in lung adenocarcinoma in the past few years, such as somatic mutations in EGFR, KRAS and EML4-ALK fusion gene. Identification of driver mutations in lung cancer is very important, since several anti-cancer drugs targeting them (gefitinib, erlotinib, and crizotinib) have shown dramatic clinical outcomes for the treatment of lung adenocarcinomas. However, a large proportion of lung cancer patients have been observed to have none of them in their cancer genome. More than 40% of lung adenocarcinoma patients appear to be driven by unknown genetic events. Gene fusions represent a subset of genetic alteration in human cancer. We recently identified a fusion gene joining KIF5B and RET transforming tyrosine kinase in tumor tissues of lung adenocarcinoma patients as a driver mutation using integration of whole-genome and transcriptome sequencing of cancer tissue [1]. We expanded our study by sequencing more transcriptomes of lung adenocarcinomas with paired normal tissues. Overall, we obtained transcriptome sequences from 88 primary lung adenocarcinomas, 53.5% (n=47) of them were negative for EGFR, KRAS and EML4-ALK. Our approach identified many novel fusion events in lung adenocarcinoma, such as CD74-ROS1, SLC34A2-ROS1, and additional fusions of cancer related genes. Majority of these fusion genes and known somatic mutations were mutually exclusive (p=0.001), suggesting these fusion genes are highly likely to be the driver mutations of lung adenocarcinoma. Overall, fusion genes account for ∼15% of genetic causes of lung adenocarcinoma according to our dataset. We will discuss those fusion genes and frequent somatic mutations of lung adenocarcinoma revealed from transcriptome sequencing of 88 lung adenocarcinomas. REFERENCE 1. Fusion of KIF5B and RET transforming gene in lung adenocarcinoma revealed from whole-genome and transcriptome sequencing. Genome Research (2012), Advance online publication Dec 22 2011.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-402. doi:1538-7445.AM2012-LB-402
Abstract LB-41: Raising awareness of ovarian cancer: a rare disease with no screening test
Authors: Leighton Susan L.
Published: August 2012

Approximately 21,000 women are diagnosed with ovarian cancer annually. Although a relatively rare disease, it is extremely difficult to detect as there is no screening test available. There are symptoms even in early stages which are dismissed by patients as not being serious and often initially being attributed to benign causes by health professional not aware of the symptom complex associated with ovarian cancer. Awareness is the only tool currently available to help improve detection. Lilies of the Valley, an ovarian cancer support and awareness group in Huntsville, Alabama, has as its mission to educate, raise awareness and disseminate information about ovarian cancer to ovarian cancer patients and survivors, the general public and the medical community in the Tennessee Valley region. The Ovarian Cancer National Alliance has its mission to remain objective in providing information to the ovarian cancer community; it does not endorse any particular treatment, therapy, clinical trial or product. These two organizations raise awareness through two very unique and innovative programs, TEAL Talks and the Survivors Teaching Students: Saving Women's Lives. This poster examines the roles of these organizations and these programs in improving early detection of ovarian cancer through patient and health professional awareness.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-41. doi:1538-7445.AM2012-LB-41
Abstract LB-421: Dual shRNA technique to screen gene-to-gene interaction
Authors: Kim Marianne K. H , Annunziata Christina
Published: August 2012

Background: Synthetic lethality screening in human cells is one of the most powerful tools to identify potential therapeutic targets in a genotype specific manner and this screening has been performed in isogenic models of matched cell-line pairs. The procedure of establishing and isolating isogenic cell lines can be arduous. And despite this laborious work, specific target(s) identified in this specific isogenic cell line may not be valid in other similar cells and experimental conditions. Therefore, we have optimized a fast and robust dual shRNA technique to screen gene-to-gene interaction. Methods and Results: To create a matched cell line pair, we utilized a bicistronic vector containing the transmembrane and extracellular domains of the mouse T-cell surface glycoprotein CD8 alpha, co-expressed with a specific shRNA, to model a specific gene deficiency. This retroviral vector was transduced into target cells, and the positively transduced cells were isolated at two days post-infection using MACS separation columns with mouse CD8a-specific microbeads. This yielded greater than 95% purity. At this time point, the decreased expression of specific gene by shRNA was minimal. After recovery from beads purification, the second bicistronic shRNA vector was introduced. This second vector contained a puromycin selectable marker with GFP and an shRNA targeting the proposed synthetic lethal gene. After second transduction, cells were selected under puromycin allowing two doubling times. The purity was 96-98% based on GFP signal measured by FACS at the end of puromycin selection. The dual-gene knockdown by sequentially introduced shRNAs achieved 70-90% depletion of each gene product. This level of knockdown was maintained for up to three weeks without significant loss of purity. After maintaining the cells in culture for several weeks CD8a purity did decrease to 70-80%, but was restored to 98-99% purity after re-applying the separation columns. The second level of purity was maintained even longer than single purification. Conclusion: In summary, we have developed a fast and efficient dual shRNA technique to apply to shRNA experimentation, obviating the need for establishing conventional isogenic cell line models. Due to its fast and simple procedure, this can be simultaneously performed in multiple cell lines. This technique can easily be adapted to screen shRNA libraries for the discovery of unknown synthetic lethal interactions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-421. doi:1538-7445.AM2012-LB-421
Abstract LB-9: Cyclooxygenase-2/15-prostaglandin dehydrogenase derived endogenous canonical NF-κB inhibitors
Authors: Snyder Nathaniel W. , Revello Sonia D. , Liu Xiaojing , Zhang Suhong , Arora Jasbir S. , Blair Ian A.
Published: August 2012

In colon, breast, and lung cancers, up-regulation of cyclooxygenase-2 (COX-2) and down-regulation of 15-prostaglandin dehydrogenase (15-PGDH) has been hypothesized to lead to a pro-proliferative, angiogenic, and chronically inflamed state. Prostaglandin E2 has been thought to drive the feed forward aspect of this pathology; however, other endogenous lipids may be involved in this process. COXs also mediate the metabolism of arachidonic acid (AA) to hydroxyeicosatetraenoic acids (HETEs), which can then be further oxidized by dehydrogenases to the corresponding keto-products. We have previously established that 11-oxo-eicosatetraenoic acid (oxo-ETE) is a major COX-2/15-PGDH derived metabolites formed from human cells expressing COX-2 treated with AA. 11-oxo-ETE has been identified from human isolates yet currently has no known receptors or known physiologic function. Earlier studies had indicated that molecules possessing a similar α,β-unsaturated ketone moiety may act as canonical Nuclear Factor-κB (NF-κB) inhibitors. Therefore, the present study was aimed to investigate the signaling of this novel COX-2/15-PGDH-derived eicosanoid. The stability and uptake of 11-oxo-ETE into human umbilical vein endothelial cells (HUVECs) was quantified with stable isotope dilution chiral liquid chromatography coupled with electron capture atmospheric pressure chemical-ionization/mass spectrometry. Tumor necrosis factor (TNF)-α induced NF-κB p50/p65 subunit nuclear translocation and consensus DNA binding was reduced by pre-incubation with 11-oxo-ETE. Dosage and reduction in NF-κB were consistent with the levels for the prostaglandin analog 15-deoxy-prostaglandin J2 (15d-PGJ2). Using a HEK293 line stably expressing a firefly luciferase reporter driven by a 5x-NF-κB consensus sequence repeat, 11-oxo-ETE and its methyl ester were shown to significantly inhibit NF-kB signaling induced by TNF-α treatment. Ethacrynic acid (EA) was used in an additional experiment to inhibit cellular glutathione-S-transferases, and this increased the ability of 11-oxo-ETE to inhibit NF-κB signaling in the reporter assay. In vitro biochemical studies also revealed inhibition of the activating kinase Inhibitor of nuclear factor-κB kinase subunit β (≥KKβ) by 11-oxo-ETE. In summary, the present study has established that the COX-2/15-PGDH derived 11-oxo-ETE can enter target endothelial cells and inhibit NF-κB p50/p65 mediated signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-9. doi:1538-7445.AM2012-LB-9
Abstract PL02-02: Lgr5 ctem cells in self-renewal and cancer
Authors: Clevers Hans
Published: August 2012

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using an inducible Cre knock-in allele and the Rosa26-LacZ reporter strain, lineage tracing experiments were performed in adult mice. The Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages throughout life, implying that it represents the stem cell of the small intestine and colon. Similar obserations were made in hair follicles and stomach epithelium.

Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids in 3D culture. Tracing experiments indicate that the Lgr5+ve stem cell hierarchy is maintained in these organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche. The same technology has now been developed for the Lgr5+ve stomach stem cells.

Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as APC. As in most cancers, the cell of origin has remained elusive. Deletion of APC in stem cells, but not in other crypt cells results in progressively growing neoplasia, identifying the stem cell as the cell-of-origin of adenomas. Moreover, a stem cell/progenitor cell hierarchy is maintained in early stem cell-derived adenomas, lending support to the “cancer stem cell”-concept.

Fate mapping of individual crypt stem cells using a multicolor Cre-reporter revealed that, as a population, Lgr5 stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. Lgr5 cell divisions occur symmetrically. The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates after cell division. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products. We find that Paneth cells are CD24+ and express EGF, TGF-a, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells dramatically improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitantloss of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents.

We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr PL02-02. doi:1538-7445.AM2012-PL02-02
Abstract PL04-02: Ras and Raf signaling: From biology to therapeutics
Authors: Marais Richard M.
Published: August 2012

The protein kinase BRAF is mutated in about 50% of human melanomas and the small G-protein NRAS is mutated in another 20% of cases. We have developed mouse models of melanoma driven by oncogenic BRAF, NRAS and KRAS. These studies have established that acquisition of BRAF, NRAS or KRAS mutations can be founder events in melanomagenesis and we are currently using these models to investigate gene-gene and gene-environment interactions in cutaneous melanoma. We are also investigating other targets of BRAF in melanoma cells and have shown that BRAF drives melanoma cell migration and invasion by down-regulating expression of the cyclic GMP phosphodiesterase PDE5A. 2011 was a landmark year in melanoma research with the registration of vemurafenib and ipilimumab, drugs that show clinical benefit in patients with advanced disease. Critically, BRAF inhibitory drugs achieve objective clinical responses in about 80% of melanomas that harbour mutations in BRAF. Surprisingly however, these drugs also drive paradoxical activation of CRAF in melanoma cells that harbour mutations in NRAS, or in cells in which RAS is activated due to increased upstream signalling. This is due to the induction of BRAF/CRAF heterodimers and CRAF homodimers and importantly, activation of the pathway by this paradox can drive tumourigenesis in some circumstances. We have used a two-stage skin carcinogenesis model to show that BRAF inhibitors are not tumour promoters pre se, but rather that they work by accelerating the growth of tumours carrying pre-existing mutations in HRAS. Thus, we conclude that BRAF inhibitors appear to accelerate the inevitable in susceptible patients, but do not induce these lesions de novo. Notably, concurrent administration of a MEK inhibitor with a BRAF drug blocks the development of these skin lesions, providing a rationale for combining BRAF and MEK inhibitors in melanoma patients. Unexpectedly, we have also found that the ABL/CKIT inhibitor nilotinib also drives paradoxical activation of the RAF pathway because it possesses weak off-target activity against BRAF and CRAF. The consequence of this paradoxical activation is that nilotinib provides a survival advantage to chronic myeloid leukaemia (CML) cells that are resistant to nilotinib, but also provides an Achilles heel to these cells because they become dependent on MEK signalling in the presence of nilotinib. Thus, MEK inhibitors cooperate with nilotinib to drive synthetic lethality in nilotinib-resistant CML cells. Thus, paradoxical activation of the RAF proteins by small molecular weight drugs can have important consequences to patient responses, creating specific clinical problems in some circumstances, but therapeutic opportunities in others.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr PL04-02. doi:1538-7445.AM2012-PL04-02
Abstract SY02-03: Bromodomain inhibition in cancer
Authors: Bradner James E.
Published: August 2012

Cellular states are maintained by coordinated gene expression programs, attributable to master regulatory transcription factors. Toward the pharmacologic control of cellular states we have undertaken a chemical biological approach to inhibit chromatin-dependent transcriptional signaling. Transcriptional activation by master regulatory proteins is facilitated by chromatin remodeling at promoter/enhancer regions, in particular side-chain acetylation of lysine residues on nearby histone tails. Context-specific lysine acetylation prompts molecular recognition by transcriptional co-activator proteins possessing acetyl-lysine recognition modules, or bromodomains. Perhaps owing to perceptions regarding the feasibility of abrogating protein-protein interactions, research toward the direct inhibition of human bromodomains (or epigenetic reader proteins, in general) has received comparatively little attention. Based on the above rationale, we have developed chemical and biochemical platforms for the development and characterization of novel bromodomain inhibitors. Recently, we reported the first potent, small-molecule inhibitor of human bromodomains, JQ1, which exhibits selectivity for the BET family of bromodomain-containing transcriptional co-activators. Of broad relevance to cancer, we will present research demonstrating that c-Myc expression is critically dependent on BRD4 function and localization to discrete upstream regulatory regions. Exposure of cancer cells to JQ1 prompts immediate down-regulation of c-Myc expression leading to suppression of a transcriptional program associated with proliferation, survival and metabolic adaptation. In translational models of Myc-dependent hematologic malignancies, the efficacy of JQ1 treatment establishes a mechanistic rationale for the leveraged clinical development of drug-like JQ1 derivatives. Toward this objective, we have completed chemical optimization of novel, drug-like inhibitors of BET bromodomains as reagents capable of supporting human clinical investigation. This research has established the feasibility of inhibiting epigenetic reader proteins with efficient, cell-permeable, small molecules. In support of an open-innovation model of chemical biology, we have created a chemical probe allowing the broad study of chromatin biology, which has already been provided to more than 150 academic, governmental and industrial laboratories worldwide. Together, we have rapidly identified mechanism-based opportunities for clinical translation in cancer, inflammation and metabolic diseases.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY02-03. doi:1538-7445.AM2012-SY02-03
Abstract SY04-01: Skin stem cells in silence, action, and cancer
Authors: Fuchs Elaine
Published: August 2012

Stem cells have the ability to self-renew long term and differentiate into one or more tissues. Typically, stem cells are used sparingly to replenish cells during normal homeostasis. This is particularly true of adult tissues which undergo relatively infrequent or periodic turnover. However, stem cells must also have the ability to respond quickly in response to injury, where they must be mobilized to undergo rapid tissue regeneration. How stem cells balance self-renewal and differentiation is of fundamental importance to our understanding of normal tissue maintenance and wound repair. Moreover, increasing evidence suggests that the regulatory circuitry governing this balancing act is at the root of some types of tumors both in mice and in humans.

The skin is an excellent model system to understand how stem cells function in normal tissue generation and how this process goes awry in cancer. Using skin as our paradigm, we've been dissecting how extrinsic signaling to stem sets off a cascade of changes in transcription that governs the activation of stem cells during tissue development, homeostasis and hair regeneration. Our findings have provided us with new insights into our understanding of the process of stem cell activation, and in so doing have revealed mechanisms which are also deregulated in a variety of different human cancers. As importantly in understanding how stem cells are activated is learning about the signals that instruct stem cells to stop making tissue.

Our recent discoveries on this topic have led us to the realm of identifying cancer stem cells (tumor-initiating cells) of squamous cell carcinomas of the skin. The second most abundant form of cancer world-wide, skin squamous cell carcinomas also establish a paradigm for many life-threatening cancers of lung, esophagus, breast, cervix, prostate, throat and oral tissues. We've demonstrated that when purified and introduced directly into the skin of a host recipient, a single cancer stem cell from a skin squamous cell carcinoma can generate a new squamous cell carcinoma that is similar in properties to the parent tumor. We show that cancer stem cell numbers and aggressiveness are predicated on the tumor microenvironment and on the ability of cancer stem cells to respond to it. The striking differences between the microenvironments of cancer stem cells and their normal counterparts seems likely to contribute to why their expression profiles are also markedly distinct. Our findings have major implications for our understanding of stem cells and cancer.

[This work was supported by the HHMI and grants from the National Institutes of Health and The Emerald Foundation]

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY04-01. doi:1538-7445.AM2012-SY04-01
Abstract SY37-02: Cell polarity proteins and cancer progression
Authors: Muthuswamy Senthil K.
Published: August 2012

The Partitioning defective protein 3 (Par3) is known to regulate asymmetric cell division, cell junction biogenesis and epithelial morphogenesis, but its role in cancer is unclear. We report that expression of Par3 is downregulated in both human breast cancers and in lung metastasis of ErbB2 transgenic mice. Downregulation of Par3 cooperated with ErbB2 to induce invasion and metastasis in vivo. Interestingly, the metastatic behavior was not associated with an overt mesenchymal phenotype. However, loss of Par3 activated a Tiam1/Rac/GTP/IRSp53/WAVE2/Arp2/3 pathway that disrupted cortical actin, blocked E-cadherin junction maturation and decreased epithelial cell-cell cohesion. Cortical actin and Arp2/3 localization were disrupted in tumor cells in vivo. Thus, we identify Par3 as a novel regulator of metastatic behavior in tumor epithelial cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY37-02. doi:1538-7445.AM2012-SY37-02
Abstract SY37-03: Targeting developmental pathways in colon cancer cells and stem cells
Authors: de Sauvage Frederic J.
Published: August 2012

The small intestine epithelium renews every 2 to 5 days, making it one of the most regenerative mammalian tissues. Genetic inducible fate mapping studies have identified two principal epithelial stem cell pools in this tissue. One pool consists of columnar Lgr5-expressing cells that cycle rapidly and are present predominantly at the crypt base. The other pool consists of Bmi1-expressing cells that largely reside above the crypt base. In the absence of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, indicating that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Interestingly, both stem cell compartments can serve as cell of origin for the development of colorectal cancer in mice. The Pathways that regulate self-renewal, commitment as well as the interplay between these two populations remain to be clearly defined but are likely to involve Wnt Notch Hedgehog. The Notch, Wnt and Hedgehog signaling pathways play critical roles during embryonic development. These factors modulate proliferation or differentiation of numerous cell types and are also involved in the regulation of the self-renewal and/or differentiation of embryonic and adult stem cells. It is however becoming increasingly clear that these pathways are involved in tumorigenesis when reactivated in adult tissues through mutations or overexpression of pathway components. Aberrant reactivation of the Hedgehog (Hh) pathway in adult tissues can lead for example to the development of cancers such as basal cell carcinoma (BCC) and medulloblastoma is often the result of inactivating mutations in PATCHED (PTCH) or activating SMOOTHENED (SMO) mutations. Notch1 mutations are found in most T-cell acute lymphoblastic leukemias and activation of the Wnt pathway, often through APC (adenomatous polyposis coli) gene mutations, is observed in a majority of colon adenocarcinomas. However all 3 pathways play a role in normal gut homeostasis and may be involved in some aspect of colon tumorigenesis.

In addition to the development of agents targeting these pathways in tumors where they are mutated we will discuss the effect on inhibiting Wnt, Notch and Hedgehog signaling on stem cells in the normal intestine and in tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY37-03. doi:1538-7445.AM2012-SY37-03