bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2180: Development of novel targeted therapies for non-small cell carcinoma by novel Midkine inhibitors.
Authors: Fukazawa Takuya , Maeda Yutaka , Hao Huifang , Yamatsuji Tomoki , Takaoka Munenori , Takigawa Nagio , Naomoto Yoshio
Published: August 2014

In the past few years a subset of driver mutations has been identified in non-small cell lung carcinoma (NSCLC), the most common type of lung adenocarcinoma. These driver mutations have differential sensitivities to various targeted therapies. The number of identified mutations in lung adenocarcinomas is limited, therefore identification of more molecular targets is needed in order to develop new treatments for NSCLCs. Efforts to find target mutations in lung adenocarcinoma with KRAS mutations are especially important because such adenocarcinomas account for 20% of lung adenocarcinoma, the most frequent type of lung adenocarcinomas.

Midkine is a heparin-binding cytokine that is induced by retinoic acid in embryonal carcinoma cells. It is involved in the development of cancer because of its mitogenic effect, promotion of angiogenesis, anti-apoptotic activity and transforming activity. Midkine expression is increased in various malignant tumors, including lung, esophageal, stomach, colon, hepatocellular, breast and pancreatic carcinomas, when compared with the level of expression in normal tissues. In a proof-of-principle effort to identify novel compounds that inhibit Midkine expression, we recently conducted cell-based high-throughput screening (HTS) of 44,000 compounds in the compound library using the Midkine promoter. We identified sixteen small molecules that inhibit Midkine promoter activity. Significantly, many of these inhibitors, which reduced Midkine protein, also inhibited proliferation in non-small cell lung cancer cells which express high levels of endogenous Midkine. We assigned the name iMDK1 to one of these compounds and assessed its ability to target non-small cell lung cancer cells. 48 hours after treatment, iMDK1 dose-dependently inhibited cell growth of Midkine expressing H441 pulmonary adenocarcinoma cells but did not impair the viability of normal proliferating human lung fibroblast cells. Hoechst 33342 staining and cell cycle analysis performed by flow cytometry demonstrated that the iMDK1 compound induced apoptosis in H441 cells. Furthermore, immunoblot analysis showed that iMDK1 inhibits the PI3 kinase pathway in H441 cells. These results indicate that iMDK1 might be a promising new therapeutic tool for the treatment of Midkine -expressing lung adenocarcinoma with KRAS mutations. Here we show a novel strategy to treat non-small cell lung cancer with Midkine inhibitors.

Citation Format: Takuya Fukazawa, Yutaka Maeda, Huifang Hao, Tomoki Yamatsuji, Munenori Takaoka, Nagio Takigawa, Yoshio Naomoto. Development of novel targeted therapies for non-small cell carcinoma by novel Midkine inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2180. doi:10.1158/1538-7445.AM2013-2180
Abstract 2187: RNAi- and U1 small nuclear interference (U1i)-mediated gene knockdown reveals the functional relevance of Pim-1 kinase in colon carcinoma and glioblastoma.
Authors: Weirauch Ulrike , Thomas Maren , Grünweller Arnold , Hartmann Roland K. , Aigner Achim M.
Published: August 2014

Pim-1 is a constitutively active serine/threonine kinase overexpressed in various tumors. Its role as proto-oncogene is based on several Pim-1 target proteins involved in pivotal cellular processes, and Pim-1 overexpression has been linked to poor prognosis.

RNAi-based knockdown approaches were used to inhibit Pim-1 in colon carcinoma cells. We demonstrate anti-proliferative, pro-apoptotic and overall antitumor effects of Pim-1 inhibition. The analysis of the molecular effects of Pim-1 inhibition reveals a complex regulatory network, with therapeutic Pim-1 repression leading to major changes in oncogenic signal transduction with regard to p21Cip1/WAF1, STAT3, JNK, c-Myc and survivin, and in the levels of apoptosis-related proteins Puma, Bax and Bcl-xL. Furthermore, Pim-1 knockdown sensitizes tumor cells towards 5-FU treatment, thereby antagonizing a 5-FU-triggered Pim-1 upregulation. This effect is mediated through decreased miR-15b levels, and our studies identify miR-15b and miR-33a to regulate Pim-1.

There is first evidence of Pim-1 overexpression also in glioblastoma, but the functional relevance is so far unknown. U1 small nuclear interference (U1i) has recently been described as novel gene silencing mechanism. It employs short oligonucleotides, so-called U1 adaptors, for specific gene knockdown. We generated Pim-1 specific U1 adaptors and demonstrate their ability to induce Pim-1 knockdown in GBM cells. U1 adaptor transfection leads to anti-proliferative and pro-apoptotic effects.

To explore therapeutic in vivo applications, we complexed siRNAs or U1 adaptors with a low molecular weight polyethylenimine (PEI) that mediates protection and cellular internalization. Treatment of tumor xenograft-bearing mice with PEI/siRNA or PEI/adaptor nanoparticles exhibits antitumor effects based on the knockdown of Pim-1, thus offering novel therapeutic strategies.

Conclusions: We demonstrate that Pim-1 plays a pivotal role in several tumor-relevant signalling pathways, and establish the functional relevance of Pim-1 in colon carcinoma and in glioblastoma. Our results also substantiate the RNAi-mediated Pim-1 knockdown based on polymeric PEI/siRNA nanoparticles as a promising therapeutic approach, and we show that U1i represents an alternative to RNAi for the therapeutic knockdown of pathologically upregulated genes.

Citation Format: Ulrike Weirauch, Maren Thomas, Arnold Grünweller, Roland K. Hartmann, Achim M. Aigner. RNAi- and U1 small nuclear interference (U1i)-mediated gene knockdown reveals the functional relevance of Pim-1 kinase in colon carcinoma and glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2187. doi:10.1158/1538-7445.AM2013-2187
Abstract 2188: A screen to identify novel p53 family member activity-restoring compounds with a focus on p73.
Authors: van den Heuvel Antonius P. , Wang Wenge , Kopelovich Levy , El-Deiry Wafik S.
Published: August 2014

Tumor suppressor p53 family regulates gene transcription, and thereby cell cycle, apoptosis, metabolism and other processes. The p53 gene is found mutated in over 50% of all human cancers, and it has been of great interest to identify drugs that can restore p53 function. While p53 can display a wide variety of mutations, which potentially could complicate function restoration therapy, family member p73 gene mutation is rare. Therefore, we sought to identify novel compounds that are able to restore the activity of the p53 family, with a focus on p73 dependency in the presence of mutant p53. To this end, a functional cell-based screening assay was set up for cancer cell death-inducing small molecules that activate p53 pathway-dependent gene transcription. SW480 colon carcinoma cells, which carry R273H/R309S mutations in p53, and stably express a p53 family-responsive luciferase construct, were utilized, with parental cells and cells displaying stable knockdown of p73 being subjected to compounds from NCI Diversity Set 3. This set contains about 1600 compounds that are relatively well characterized and structurally rigid. After various treatment periods, p53 activity was assessed, as well as cell viability. Subsequently, a total of 26 compounds were selected for further investigation and validation. Follow up studies will include dose and time courses for p53 family activation and viability in a variety of cell lines that are wild-type, mutant or null for p53. Also mechanistic studies and cellular assays will be performed, with the hopes to identify novel drugs that are able to restore activity of the p53 family, and subsequent cancer cell death, and thus providing novel candidates for the treatment of human cancer.

Citation Format: Antonius P. van den Heuvel, Wenge Wang, Levy Kopelovich, Wafik S. El-Deiry. A screen to identify novel p53 family member activity-restoring compounds with a focus on p73. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2188. doi:10.1158/1538-7445.AM2013-2188
Abstract 2190: cIAP2 as a therapeutic target in malignancies.
Authors: Miura Koh , Karasawa Hideaki , Fujibuchi Wataru , Ohnuma Shinobu , Unno Michiaki
Published: August 2014

[Introduction] Inhibition of the apoptotic pathway is one of the factors that may be responsible for carcinogenesis and drug resistance, and the inhibitor of apoptosis protein (IAP) family is thought to prevent apoptosis through inhibition of caspases and pro-caspases and may affect anti-cancer drug sensitivity. [Purposes] The purpose of this study is to clarify the roles of cellular inhibitor of apoptosis 2 (cIAP2) gene, which is a member of IAP family, on apoptosis and 5-FU sensitivity in cancer cells. [Methods and Results] The expression profiles of human colon cancer cell line DLD-1, its 5-FU-resistant subclone DLD-1/FU and further 21 types of colon cancer cell lines were compared. 50% inhibitory concentration (IC50) of 5-FU in DLD-1/FU was over 100 times higher than that in DLD-1. With the analysis of expression profiles among 23 genes, we found that cIAP2 gene was up-regulated in DLD-1/FU compared with DLD-1 and other cell lines with low IC50. Next, the role of the cIAP2 gene was investigated for 5-FU resistance using RNA interference. The down-regulation of cIAP2 efficiently enhanced 5-FU sensitivity, the activation of caspase 3/7 and apoptosis under exposure to 5-FU. The immunohistochemistry of cIAP2 in cancer and corresponding normal tissues from colorectal cancer patients in stage III revealed that cIAP2 was more frequently expressed in cancer tissues than in normal tissues, and cIAP2-positive patients had a trend toward early recurrence after 5-FU-based chemotherapy. [Conclusion] IAPs are reported to be abnormally regulated and expressed in the majority of human malignancies at elevated levels. Among them, cIAP2 should be a possible therapeutic target in malignancies, especially in colorectal cancer.

Citation Format: Koh Miura, Hideaki Karasawa, Wataru Fujibuchi, Shinobu Ohnuma, Michiaki Unno. cIAP2 as a therapeutic target in malignancies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2190. doi:10.1158/1538-7445.AM2013-2190
Abstract 2192: A novel mechanism of EML4-ALK rearrangement in a patient-derived cell line and its tumor growth inhibition by an ALK inhibitor CH5424802.
Authors: Kodama Tatsushi , Motoi Noriko , Ninomiya Hironori , Sakamoto Hiroshi , Kitada Kunio , Tsukaguchi Toshiyuki , Sato Yasuko , Ishii Nobuya , Terui Yasuhito , Hatake Kiyohiko , Ishikawa Yuichi
Published: August 2014

Purpose: EML4-ALK is a driver oncogene in non-small cell lung cancer (NSCLC) and has been developed into a promising molecular target for antitumor agents. Although EML4-ALK is reported to be formed by inversion of chromosome 2, other mechanisms of gene fusion remain unknown. Here we characterize the EML4-ALK fusion gene of a new cell line derived from a patient of NSCLC. In addition, the efficacy of ALK inhibitors against this new cell line was examined.

Experimental Design: An EML4-ALK-positive cell line, termed LC649, was established from pleomorphic carcinoma, a rare subtype of NSCLC. We investigated the chromosomal aberrations using fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). CH5424802, a selective ALK inhibitor, was evaluated in the antitumor activity against LC649 in vitro and in vivo xenograft model.

Results: Our initial characterization revealed that LC649 harbors variant 3 of EML4-ALK fusion with 2-fold copy number gain. Interestingly, CGH and metaphase-FISH analysis showed that LC649 contained, in addition to two normal chromosome 2, two aberrant chromosome 2 that were fragmented and scattered. These observations provided the first evidence that EML4-ALK fusion in LC649 cells was formed in chromosome 2 by a distinct mechanism of genomic rearrangement, termed “chromothripsis”. Additionally, CH5424802 inhibited phospho-ALK and suppressed tumor growth of the LC649 cells in vitro and in vivo in a xenograft model.

Conclusions: Our results suggested that chromothripsis may be a mechanism of oncogenic rearrangement of EML4-ALK. CH5424802 was effective against EML4-ALK positive tumors with ALK copy number gain mediated by chromothripsis.

Citation Format: Tatsushi Kodama, Noriko Motoi, Hironori Ninomiya, Hiroshi Sakamoto, Kunio Kitada, Toshiyuki Tsukaguchi, Yasuko Sato, Nobuya Ishii, Yasuhito Terui, Kiyohiko Hatake, Yuichi Ishikawa. A novel mechanism of EML4-ALK rearrangement in a patient-derived cell line and its tumor growth inhibition by an ALK inhibitor CH5424802. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2192. doi:10.1158/1538-7445.AM2013-2192
Abstract 2193: Characterization of the aggresome targeted Q141K ABCG2/BCRP variant.
Authors: Basseville Agnes , Shukla Suneet , Tamaki Akina , Robey Robert , Ambudkar Suresh V. , Bates Susan E.
Published: August 2014

ABCG2 is a member of the G subfamily of human ABC transporters. It is an ATP-dependent protein and is a half-transporter that must dimerize for function. In various tumor cell lines, ABCG2 was involved in resistance to chemotherapy, by transporting numerous anticancer agents including tyrosine kinase inhibitors. Studies show the transporter has an important role in normal tissue protection, including the blood-brain and maternal-fetal barriers. It has also been observed that ABCG2 may play a protective role with respect to exposure to smoke or dietary carcinogens. A common variant in human ABCG2, the polymorphism Q141K, impairs expression and function, thereby reducing drug clearance and increasing the toxicity of chemotherapy and other substrate drugs. Although the activity of the Q141K variant is regularly described, biochemical data remain rare. Q141K has an impaired trafficking to cell surface compared to the WT protein and is sequestered in the aggresome. Here we characterized the Q141K and WT protein in human cells, studying post-translational modification, biochemistry, dimerization status, half-life, and endocytosis. Glycosylation and phosphorylation are equivalent, as is ATP binding and hydrolysis, and substrate binding using IAAP competition assays. Cell treatment with romidepsin, an HDAC inhibitor approved by FDA as an anticancer agent, restored the impaired variant trafficking, leading to an increase in drug efflux. The restoration of ABCG2 Q141K function has potential applications in oncology and in medicine, in improving normal tissue protection and drug elimination, as well as in cancer prevention since several carcinogens are substrates for ABCG2.

Citation Format: Agnes Basseville, Suneet Shukla, Akina Tamaki, Robert Robey, Suresh V. Ambudkar, Susan E. Bates. Characterization of the aggresome targeted Q141K ABCG2/BCRP variant. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2193. doi:10.1158/1538-7445.AM2013-2193
Abstract 2194: Tyrosyl DNA phosphodiesterase I is a prognostic factor and its inhibition synergizes response to topoisomerase poisons in malignant glioma.
Authors: Al-Keilani Maha , Agarwal Supreet , Alqudah Mohammad , Sibenaller Zita , Ryken Timothy , Assem Mahfoud
Published: August 2014

Malignant Glioma is the most common type of primary brain tumors in adults accounting for about 80% of all gliomas. Despite the advances in treatment and diagnosis, the prognosis of malignant glioma is still poor with a median survival less than a year for glioblastoma patients. Targeting DNA-repair mechanisms is a clinically relevant option for enhancing response to DNA-damaging anticancer agents. TDP1 has been recently discovered to be responsible for the repair of the irreversible DNA-topoisomerase I covalent complexes produced by topoisomerase I poisons. Other studies have suggested its role in the repair of the DNA damage produced by ionizing radiation and temozolomide. However, TDP1 relevance has not yet been studied in glioma. Thus the aims of this project are to identify the role of TDP1 in glioma aggressiveness and resistance to therapies. We hypothesize that high-grade gliomas possess higher TDP1 levels which are associated with lower survival and resistance to therapy.

Quantitative real-time PCR analysis of TDP1 in glioma biopsies revealed a negative prognostic effect of TDP1, where the level of TDP1 mRNA was inversely correlated with patient survival. Moreover, western blot showed an overexpression of TDP1 in glioma biopsies compared to normal tissues and increased expression with higher grades. TDP1 overexpression in U87 cell lines did not induce resistance to temozolomide, topotecan, etoposide or doxorubicin. This may be related to other downstream DNA repair enzymes that need to be also modulated to fully induce drug resistance. However, TDP1 inhibition using small molecules synergizes the cytotoxicity of topotecan, etoposide, and doxorubicin, confirming thus its critical role in response to several therapeutic agents.

As a conclusion, TDP1 is a new and promising prognostic factor and a modulator of therapy response in malignant glioma that may help in stratification of patients. Additionally, targeting TDP1 is a novel strategy that will widen the therapeutic options for malignant glioma where topoisomerase I and II poisons can be part of the gold standard therapy when used in combination with TDP1 inhibitors.

Citation Format: Maha Al-Keilani, Supreet Agarwal, Mohammad Alqudah, Zita Sibenaller, Timothy Ryken, Mahfoud Assem. Tyrosyl DNA phosphodiesterase I is a prognostic factor and its inhibition synergizes response to topoisomerase poisons in malignant glioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2194. doi:10.1158/1538-7445.AM2013-2194
Abstract 2197: p53-dependent expression of the fluorouracil (5-FU) catabolic enzyme dihydropyrimidine dehydrogenase (DPD) in mouse liver.
Authors: Gokare Prashanth R. , Finnberg Niklas , Allen Joshua , Dai Jenny , El-Deiry Wafik
Published: August 2014

Fluorouracil (5-FU) a commonly used drug in the treatment of cancer is hampered by a short unpredictable plasma half-life, which complicates dose modulation to minimize toxicity and improve efficacy of 5-FU treatment. Strategies to improve on the unfavorable pharmacokinetic properties of 5-FU, currently under clinical trial, involve targeting of the catabolic enzyme dihydropyrmidine dehydrogenase (DPD).This requires understanding the mechanisms regulating the expression of DPD and the inducibility of 5-FU catabolism, which is inadequately investigated. By performing in silico analysis we were able to identify putative p53 binding sites in the proximity of the dihydropyrimidine dehydrogenase gene (DPYD) and confirm by chromatin immunoprecipitation (ChIP) that p53 increasingly locates to a binding site (about 1.8 ± 0.05 fold over untreated control) downstream of DPYD (P53DBS) following a single IV bolus of 5-FU (150 mg/kg bw) in mouse liver. Indeed, DPYD mRNA increased by 1.8 fold and protein by 1.8 fold (P< 0.0005) respectively in a time- and p53-dependent manner in the liver of treated with 5-FU. Our ongoing studies are focused on assessing p53-dependent regulation of DPD-activity and the resulting change in 5-FU bioavailability We suggest that p53 can regulate an important step of pyrimidine-catabolism and targeting p53 in the context of 5-FU therapy of malignancies carrying p53 mutations may be another strategy to improve treatment efficacy.

Citation Format: Prashanth R. Gokare, Niklas Finnberg, Joshua Allen, Jenny Dai, Wafik El-Deiry. p53-dependent expression of the fluorouracil (5-FU) catabolic enzyme dihydropyrimidine dehydrogenase (DPD) in mouse liver. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2197. doi:10.1158/1538-7445.AM2013-2197
Abstract 2203: Characterization of raloxifene glucuronidation and the role of UGT genotypes on raloxifene metabolite profiles in vivo.
Authors: Sun Dongxiao , Jones Nathan R. , Andrea Manni , Lazarus Philip
Published: August 2014

Raloxifene is a 2nd-generation selective estrogen receptor modulator used for the prevention and treatment of osteoporosis and the prevention of breast cancer in postmenopausal women. Raloxifene is extensively metabolized by glucuronidation to form raloxifene-6-glucuronide (ral-6-Gluc) and raloxifene-4’-glucuronide (ral-4’-Gluc). The goal of the present study was to determine whether functional polymorphisms in active UGTs could play a role in altered raloxifene glucuronidation in vivo. Using homogenates from HEK293 UGT-overexpressing cell lines, raloxifene was shown to be glucuronidated primarily by the hepatic UGTs 1A1 and 1A9 and the extra-hepatic UGTs 1A8 and 1A10; no detectable raloxifene glucuronidation activity was found for UGT2B enzymes. Functional UGT1A1 transcriptional promoter genotypes were significantly (ptrend=0.005) associated with ral-6-Gluc formation in human liver microsomes, and, consistent with the decreased raloxifene glucuronidation activities observed in vitro with cell line over-expressing UGT1A8 variants, the UGT1A8*2 variant was significantly (p=0.023) correlated with total raloxifene glucuronide formation in human jejunum homogenates. While ral-4’-Gluc exhibited 1/100th the anti-estrogenic activity of raloxifene itself as measured by binding to the estrogen receptor, raloxifene glucuronides comprised ∼99% of the circulating raloxifene dose in raloxifene-treated subjects, with ral-4’-Gluc comprising ∼70% of raloxifene glucuronides. Plasma ral-6-Gluc (ptrend=0.0025), ral-4’-Gluc (ptrend=0.001), and total raloxifene glucuronides (ptrend=0.001) were increased in raloxifene-treated subjects who were predicted slow metabolizers [UGT1A8 (*1/*3)] vs intermediate metabolizers [UGT1A8 (*1/*1) or UGT1A8 (*1/*2)] vs fast metabolizers [UGT1A8 (*2/*2). These data suggest that raloxifene metabolism may be dependent on UGT1A8 genotype and that UGT1A8 genotype may play an important role in overall response to raloxifene.

Citation Format: Dongxiao Sun, Nathan R. Jones, Manni Andrea, Philip Lazarus. Characterization of raloxifene glucuronidation and the role of UGT genotypes on raloxifene metabolite profiles in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2203. doi:10.1158/1538-7445.AM2013-2203
Abstract 2209: Contribution of FKBP5 genetic variation to gemcitabine treatment and survival in pancreatic cancer.
Authors: Ellsworth Katarzyna A. , Eckloff Bruce W. , Li Liang , Moon Irene , Fridley Brooke L. , Jenkins Greg D. , Carlson Erin , Brisbin Abra , Abo Ryan , Bamlet William , Petersen Gloria , Wieben Eric D. , Wang Liewei
Published: August 2014

The FKBP5 gene encodes the FKBP51 immunophilin, a negative regulator of AKT. Variability in level of FKBP5 expression is a major factor contributing to variation in response to several classes of chemotherapeutic agents including gemcitabine, first line treatment for pancreatic cancer. Genetic variation of FKBP5 could influence its expression, function, and, ultimately, treatment response of pancreatic cancer. We set out to comprehensively study the role of genetic variation in FKBP5 identified by Next Generation DNA resequencing in response to gemcitabine treatment of pancreatic cancer by utilizing both tumor and germline DNA samples from 43 pancreatic cancer patients, including 19 paired tumor-normal samples. Genotype-phenotype association studies with overall survival (in tumor only) as well as with FKBP5 gene expression were performed using the same samples in which Next Generation DNA resequencing had been performed, followed by functional genomic studies of top selected single nucleotide polymorphisms (SNPs). In-depth resequencing identified 404 FKBP5 SNPs in both normal and tumor DNA patient samples. SNPs with the smallest p-values for association with FKBP5 survival or expression were subjected to functional genomic studies. Electromobility shift assay (EMSA) showed that the rs73748206 “T” allele altered DNA-protein binding patterns, consistent with a significantly increased reporter gene activity. The effect of rs73748206 was also confirmed by mRNA expression studies using lymphoblastoid cell lines (LCL) derived from the same patients from whom DNA used for resequencing had been obtained. This comprehensive FKBP5 resequencing study provides insight into the role of inheritance in variation in response to gemcitabine therapy of pancreatic cancer.

Citation Format: Katarzyna A. Ellsworth, Bruce W. Eckloff, Liang Li, Irene Moon, Brooke L. Fridley, Greg D. Jenkins, Erin Carlson, Abra Brisbin, Ryan Abo, William Bamlet, Gloria Petersen, Eric D. Wieben, Liewei Wang. Contribution of FKBP5 genetic variation to gemcitabine treatment and survival in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2209. doi:10.1158/1538-7445.AM2013-2209
Abstract 221: Cancer stem cell-selected patient-derived xenograft models with sensitivity to FAK and Pan-PI3K/mTOR inhibitors .
Authors: Weaver David T. , Paterson Daniel , Padval Mahesh , Pachter Jonathan , Lewis Michael
Published: August 2014

In this study, Tumor-initiating (Creighton et al, 2009) and EMT gene expression signatures (Gupta et al., 2009, Taube et al, 2009) were used as classifiers. Tumors from 25 human triple negative breast cancer Patient-Derived Xenografts (PDX) were examined with Affymetrix U133Plus2 RNA arrays. Datasets were RMA normalized, and samples scored with the TI or EMT signature based on the Pearson Correlation Coefficient of a vector of mean-centered expression values of signature genes and a vector of indicators for signature genes such that 1 (up-regulated, correlated) genes and -1 (down-regulated, anti-correlated) genes formed a TI or EMT score. A high EMT score is also representative of mesenchymal tumors. Several of the triple negative breast cancer PDX models had both high TI and high EMT scores, indicated by p<0.05 of a one-sided Pearson Correlation Coefficient test. Two of the PDX models with high positive TI and EMT scores, 4664 and 3887, were evaluated for sensitivity to a FAK inhibitor (VS-4718), a pan-PI3K/mTOR inhibitor (VS-5584), and chemotherapy (docetaxel). FAK and PI3K/mTOR are implicated in tumorigenesis, including tumor initiation, growth, angiogenesis and metastasis. Both 4664 and 3887 tumors were N-cadherin-positive (mesenchymal), and strongly positive for CD44 (TI marker) by immunohistochemistry. These tumors were also weakly positive for E-cadherin and highly heterogeneous for pan-cytokeratin, two epithelial marker patterns suggesting a mesenchymal phenotype. Whereas 3887 was Notch3-positive, 4664 was Notch3-negative. VS-4718-treated 4664 and 3887 models showed diminished tumor growth over 21 days compared with vehicle-treated animals. VS-4718-treated animal tumors showed decreased FAK autophosphorylation (pY397) by immunohistochemistry. Likewise, the VS-5584-treated 4664 and 3887 animals blocked tumor growth over 21 days. In these models, the PI3K/mTOR pathways activate downstream signaling phosphorylation of pS6 pSer240/244, p4EBP1 pThr70, and pPRAS40 pThr246. In the VS-5584-treated 3887 tumors, pS6 pSer240/244 levels were significantly diminished relative to vehicle- and docetaxel-treated tumors. As predicted by TI and EMT gene expression indicators, both models were docetaxel-resistant.

These findings and our previous work on these agents for the inhibition of CSCs support the clinical development of the FAK and the pan-PI3K/mTOR inhibitors to target CSCs for treatment of breast, mesothelioma, ovarian and other cancers.

Citation Format: David T. Weaver, Daniel Paterson, Mahesh Padval, Jonathan Pachter, Michael Lewis. Cancer stem cell-selected patient-derived xenograft models with sensitivity to FAK and Pan-PI3K/mTOR inhibitors . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 221. doi:10.1158/1538-7445.AM2013-221
Abstract 2218: Systematic identification of combinatorial markers of drug sensitivity in cancer cell lines.
Authors: Tabchy Adel , Eltonsy Nevine , Mills Gordon B.
Published: August 2014

Purpose: Translating the cancer genome into highly efficacious targets to guide rational therapeutic combinations is a major emerging challenge.

Methods: We established an in silico bioinformatic platform in parallel with a high throughput screening platform evaluating the pharmacological activity of 37 novel targeted agents across 669 highly characterized cell lines representing the genetic and tumor-type heterogeneity of human cancers. Analysis of large scale pharmacological data coupled to massive sequencing data on cell lines was performed to systematically identify combinatorial biomarkers of sensitivity and resistance to cancer therapeutics. Genomic predictors discovered in a 141 cell line training set were validated in an independent non-overlapping test set of 359 cell lines screened on 14 of the compounds.

Results: We demonstrate combinations of genomic events that are co-occurring or mutually exclusive and act as co-drivers in various tumors, representing potential targets for combinatorial intervention in cancer. We find that multiple cooperating genomic events predict response to drug intervention independent of tumor lineage.

Conclusions: The coupling of scalable in silico and functional high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to prevent the emergence of resistance.

Citation Format: Adel Tabchy, Nevine Eltonsy, Gordon B. Mills. Systematic identification of combinatorial markers of drug sensitivity in cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2218. doi:10.1158/1538-7445.AM2013-2218
Abstract 2220: Toxicities and antitumor efficacy of tumor-targeted AuNRs in mouse model.
Authors: Peng Xianghong , Mackey Megan , Austin Lauren , Oyelere Adegboyega , Chen Georgia , Huang Xiaohua , El-Sayed Mostafa A. , Shin Dong M.
Published: August 2014

Near infrared (NIR)-absorbing gold nanorods (AuNRs) can be systemically or locally delivered into tumors and subsequent irradiated by nonionizing NIR laser light, and creates heat to destroy cancer cells, this technique has been demonstrated to dramatically improve the efficacy of conventional plasmonic photothermal therapy (PPTT) without contrast agents or with molecular agents of dye molecules. In the present study, we have synthesized new HNSCP-1-conjugated AuNRs for targeting the squamous cell carcinoma of the head and neck (SCCHN), and studied the biodistribution and associated toxicity of such SCCHN-targeted AuNRs in a BALB/c mouse model and SCCHN xenograft models. Our results showed that the conjugation of HNSCP peptide to AuNRs decreased the blood circulation time compared with that of the PEG-coated AuNRs (22nm). The PEG coated-AuNRs (10,000/AuNR, 22nm) were mainly found in the liver(57%) and spleen (38%), while about 92% of the injected HNSCP-1 peptide conjugated-AuNRs (10,000/AuNR) accumulated in the liver (72 hours after AuNRs injection). The tumor-targeted AuNRs increased the accumulation of AuNRs in the tumor mass compared with that of non-targeted AuNRs. The long-term uptake fate of the AuNRs in a mouse model (15 months after injection of 3.6 μg AuNRs) has been studied, and no remarkable toxicities have been observed. Our results showed that the PPTT treatments (single dose of intratumoral injection of 0.5ug HNSCP-1-AuNRs, followed by 2 min of 0.9-1.1 W/cm2 NIR laser exposure) could inhibit the tumor growth in nude mice bearing human SCCHN Tu212 xenograft tumor model. Conclusions: HNSCP-1-AuNRs may selectively accumulate in the tumor mass and inhibit the tumor growth via the PPTT without observed toxicities.

This research was supported by NCI U01 CA 151802.

Citation Format: Xianghong Peng, Megan Mackey, Lauren Austin, Adegboyega Oyelere, Georgia Chen, Xiaohua Huang, Mostafa A. El-Sayed, Dong M. Shin. Toxicities and antitumor efficacy of tumor-targeted AuNRs in mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2220. doi:10.1158/1538-7445.AM2013-2220
Abstract 2221: Enhanced SH3:linker interaction allosterically sensitizes Abl kinases to small molecule inhibitors.
Authors: Panjarian Shoghag B. , Iacob Roxana , Shugui Chen , Wales Thomas , Engen John R. , Smithgall Thomas E.
Published: August 2014

Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for the pathogenesis of chronic myelogenous leukemia (CML). Clinical management of CML has been revolutionized by imatinib, a selective ATP-competitive inhibitor of Bcr-Abl kinase activity. Imatinib selectivity is due in part to its ability to recognize and stabilize the inactive conformation of the kinase domain. This conformation is stabilized by intramolecular binding of the SH3 domain to the

SH2-kinase linker, which in turn packs against the back of the kinase domain. Imatinib resistance can be caused by point mutations that either affect drug binding directly (e.g., through mutation of the gatekeeper position, Thr315) or that allosterically disturb the downregulated kinase domain conformation (e.g., through mutation or phosphorylation of the SH3 domain). These observations suggest that strengthening the regulatory SH3:linker interaction present in

Bcr-Abl may stabilize a downregulated kinase domain conformation, thus sensitizing the kinase domain to inhibitor action. To explore this possibility, we created a series of modified c-Abl and Bcr-Abl proteins with enhanced

SH3-linker interactions. By systematically increasing the proline content of the linker, we identified high-affinity linkers that stabilized intramolecular

SH3 binding without disturbing the overall regulation of the kinase core.

Enhanced SH3:linker interaction completely reversed c-Abl core activation by a

C-lobe myristate binding pocket mutation, and substantially reduced activation by mutations in the ATP binding site (T315I) as well as the SH2:C-lobe interface (Y158D). Remarkably, enhanced SH3:linker interaction dramatically sensitized Bcr-Abl not only to imatinib but also to the myristic acid binding pocket inhibitor, GNF-2. These effects were observed in the context of both wild-type and imatinib-resistant forms of Bcr-Abl. Hydrogen exchange mass spectrometry of recombinant Abl core proteins with high-affinity linkers revealed dynamic coupling between the SH3:linker interface, the GNF-2 binding site in the C-lobe myristate binding pocket, and the active site.

Taken together, these studies provide strong evidence that regulatory

SH3:linker interaction is retained in the context of Bcr-Abl, and supports the idea that small molecules enhancing natural regulatory interaction at the

SH3:linker interface may have utility as chemical sensitizers of Bcr-Abl drug action in the kinase domain.

Citation Format: Shoghag B. Panjarian, Roxana Iacob, Chen Shugui, Thomas Wales, John R. Engen, Thomas E. Smithgall. Enhanced SH3:linker interaction allosterically sensitizes Abl kinases to small molecule inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2221. doi:10.1158/1538-7445.AM2013-2221
Abstract 2224: Re-engineering of the duocarmycin structural architecture enables tumour-selective CYP2W1-mediated drug activation in human colon cancer xenografts.
Authors: Pors Klaus , Travica Sandra , Loadman Paul M. , Shnyder Steven D. , Sutherland Mark , Sheldrake Helen M. , Searcey Mark , Johansson Inger , Mkrtchian Souren , IngelmanSundberg Magnus , Patterson Laurence H.
Published: August 2014

There is now growing evidence that CYP1A1, 1B1, 2J2, 2S1 and 2W1 are over-expressed in many human tumour types. The ultrapotent duocarmycins are ideal candidates for bioprecursor drug development and we have demonstrated that these can be re-engineered into derivatives selectively activated by CYP1A1 in vitro and in vivo.1,2 In the present study, we have focussed on CYP2W1 as a potential therapeutic target. CYP2W1 is detected in 30% of colon cancers, while its protein expression in non-transformed adult tissues is absent or insignificant.3 We have presented results indicating that the immunohistochemically assessed level of CYP2W1 enzyme could fulfil the criteria for being a prognostic biomarker for stages II and III colorectal cancer.4 Here we present data on furanoindole-based duocarmycins that have the potential to be used as a chemical probe (e.g. ICT2726) to show CYP2W1 functional activity. We also report on indoline-based analogues that elicit potent antiproliferative activity after CYP2W1-mediated activation in cancer cells. Specifically, we have transfected human HEK293, Colo320 and SW480 cells to express CYP2W1 and shown that these suffer rapid loss of viability following treatment with two halogenated duocarmycin bioprecursor compounds (ICT2705 and ICT2706) while no activity in mock-transfected cells was seen. Significantly, ICT2706 was shown to totally prevent tumour growth when administered to SCID mice bearing SW480-2W1 xenografts (dosed daily with 100 mg/kg for 8 days) but not the mock-transfected variant. Subsequent to this treatment, the tumours were extracted for further analysis. Using H2A.X phosphorylation as a marker for DNA damage, our data revealed a time-dependent increase in expression supporting CYP2W1-mediated activation of ICT2706 in vivo. Our findings reveal the opportunities in targeting CYP2W1 as a novel therapeutic approach in colon cancer chemotherapy.

Citation Format: Klaus Pors, Sandra Travica, Paul M. Loadman, Steven D. Shnyder, Mark Sutherland, Helen M. Sheldrake, Mark Searcey, Inger Johansson, Souren Mkrtchian, Magnus IngelmanSundberg, Laurence H. Patterson. Re-engineering of the duocarmycin structural architecture enables tumour-selective CYP2W1-mediated drug activation in human colon cancer xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2224. doi:10.1158/1538-7445.AM2013-2224
Abstract 2225: The molecular architecture of p85α as determined by SAXS and chemical cross-linking.
Authors: Brower Evan T. , Gabelli Sandra B. , Wang Qing , Chaerkady Raghothama , Berndsen Christopher E. , Cole Robert N. , Backer Jonathan M. , Schäfer Mathias , Sinz Andrea , Kinzler Kenneth W. , Vogelstein Bert , Amzel L. Mario
Published: August 2014

The p85α protein, encoded by PIK3R1, is the regulatory subunit of phosphatidylinositol 3-kinase alpha (PI3Kα). The role of p85α extends to regulation of PTEN-phosphatase activity, the unfolded protein response, and additional cellular pathways. PIK3R1 was recently identified as frequently mutated in glioblastomas and endometrial cancers. Small-angle X-ray scattering (SAXS) and chemical cross-linking were used to complete the first investigation of the shape and molecular organization of full length p85α. p85α is elongated in shape and exists in a conformation conducive to p110α binding. The p85α structural data was utilized to generate the first model of the full length PI3K complex. Cancer associated PIK3R1 mutations are localized to interfaces between p85α domains, and in the context of the PI3K complex, are clustered at regions comprising the interface of the catalytic and regulatory subunits. A potential interface was discovered between the RhoGAP domain of p85α and the p110α kinase domain. GTPases may enhance PI3K activity by binding to the RhoGAP domain, thereby attenuating the interaction between the RhoGAP and the kinase domain. These data provide the structural groundwork required for a better understanding of the regulatory mechanisms mediated by p85, and may facilitate the development of future drugs targeting the PI3K pathway.

Citation Format: Evan T. Brower, Sandra B. Gabelli, Qing Wang, Raghothama Chaerkady, Christopher E. Berndsen, Robert N. Cole, Jonathan M. Backer, Mathias Schäfer, Andrea Sinz, Kenneth W. Kinzler, Bert Vogelstein, L. Mario Amzel. The molecular architecture of p85α as determined by SAXS and chemical cross-linking. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2225. doi:10.1158/1538-7445.AM2013-2225
Abstract 2227: Aptamer selection for cancer markers: High-throughput, quantitative selection and characterization of nucleic acid aptamers for human angiopoietin-2.
Authors: Cho Minseon , Oh Seung Soo , Nie Jeff , Stewart Ron , Eisenstein Michael , Chambers James , Marth Jamey D. , Thomson James A. , Soh Tom H.
Published: August 2014

Abstract

Nucleic acid-based aptamers represent a promising class of affinity reagents, as they can be chemically synthesized and modified, undergo reversible folding and exhibit excellent stability. Thus, the identification of aptamers that bind to specific cancer markers may serve as a powerful tool for early diagnosis and treatments of cancers. Unfortunately, conventional methods of aptamer generation remain inherently low-throughput in nature, and there is a pressing need for alternative technologies that can generate high quality aptamers in a high-throughput and economical manner.

Towards the goal of accelerating aptamer discovery, we have developed the Quantitative Parallel Aptamer Selection System (QPASS) - an integrated platform that combines microfluidic selection, next-generation sequencing and in situ-synthesized aptamer arrays for parallel binding measurements. As a model, we used QPASS to select aptamers that specifically bind to human angiopoietin-2, an important mediator of angiogenesis and a biomarker of colon, prostate and breast cancers; after microfluidic selection and sequencing, we fabricated a custom aptamer array containing 120,000 sequences representing the most enriched aptamer candidates. Using this array, we simultaneously obtained equilibrium dissociation constants (Kd) and binding specificity data for these candidate aptamers in a massively parallel manner. Furthermore, we show that our array can operate directly in undiluted serum, enabling rapid identification of aptamers that perform optimally in complex mixtures. By exploiting the scalability and commercial availability of high-throughput sequencing and nucleic acid arrays, the QPASS platform offers the capability to simultaneously analyze and characterize unprecedented numbers of aptamer candidates—either against a common cancer marker, or for screening in parallel against multiple different biomarkers.

Citation Format: Minseon Cho, Seung Soo Oh, Jeff Nie, Ron Stewart, Michael Eisenstein, James Chambers, Jamey D. Marth, James A. Thomson, Tom H. Soh. Aptamer selection for cancer markers: High-throughput, quantitative selection and characterization of nucleic acid aptamers for human angiopoietin-2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2227. doi:10.1158/1538-7445.AM2013-2227
Abstract 2228: Phosphatidylinositol-4-kinase - Potent and selective inhibitors of PI4Kα and PI4Kβ.
Authors: Waring Michael J. , Cross Darren , Andrews David , Flemington Vikki , Lenaghan Carol , McKelvie Jennifer , Maman Sarita , Raubo Piotr , Robb Graeme , Smith James , Swarbrick Martin
Published: August 2014

Phosphatidylinositol (PI) is a phospholipid that resides primarily on the cytosolic surface of cell membranes. PI can be phosphorylated to generate seven different phosphatidylinositol phosphates (PIPs), all of which have distinct biological activities.1 A major first step in the production of these PIPs is the conversion of PI into phosphatidylinositol-4-phosphate (PI4P) by PI4 kinase.2

Here we describe the identification of inhibitors of PI4 kinase via targeted screening of a kinase subset. This exercise has delivered inhibitors that are potent inhibitors of PI4 kinase (pIC50 >8), which are selective the α- or the β-subtypes and do not inhibit related enzymes on the PI pathway (PI3 kinase pIC50 <5 or PIP5 kinase pIC50 <4) and also exhibit good broader kinase selectivity.

The effect of these useful probe compounds on cellular phosphatidylinositol-4,5-diphosphate and subsequent downstream markers will be described along with the comparison of these effects with the corresponding siRNA.

1. G. Di Paolo, P. De Camilli, Phosphoinositides in cell regulation and membrane dynamics. Nature 2006, 443, 651-657.2. T. Sasaki, S. Takasuga, J. Sasaki, S. Kofuji, S. Eguchi, M. Yamazaki, A. Suzuki, Mammalian phosphoinositide kinases and phosphatases. Progress in lipid research, 2009, 48, 307-343.

Citation Format: Michael J. Waring, Darren Cross, David Andrews, Vikki Flemington, Carol Lenaghan, Jennifer McKelvie, Sarita Maman, Piotr Raubo, Graeme Robb, James Smith, Martin Swarbrick. Phosphatidylinositol-4-kinase - Potent and selective inhibitors of PI4Kα and PI4Kβ. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2228. doi:10.1158/1538-7445.AM2013-2228