bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4285: Intra-individual variation in circulating miRNA expression levels in human plasma samples
Authors: Wu Jie , Cai Hui , Xiang Yong-Bing , Matthews Charles , Ye Fei , Zheng Wei , Cai Qiuyin , Shu Xiao-Ou
Published: July 2016

Circulating miRNAs as possible non-invasive biomarkers for disease risk assessment and cancer early diagnosis has attracted increasing interest. Little information, however, is available regarding the intra-individual variation of circulating miRNA levels. We measured miRNA expression in repeated plasma samples that were collected 6 to 12 months apart from 53 healthy individuals participating in a sub-study nested in the Shanghai Women's and Shanghai Men's Health Studies. Total RNA, including miRNA, was isolated from plasma using Qiagen's miRNeasy Serum/Plasma Kit. Three synthetic spike-in RNA oligos (osa-miR414, cel-miR248, and ath-miR159a) were added to control for variances in the starting material and the efficiency of RNA extraction. NanoString's Human v2 miRNA Expression Assay, which includes CodeSets of probes specific for 800 common human miRNAs, was used for the miRNA assays. The derived miRNA counts were first corrected by subtracting the background counts and then were normalized by using the average count from three spike-in oligos and

the top fifty miRNAs that gave most significant hybridization signals. The intra-individual variation was evaluated by an intra-class correlation coefficient (ICC). A total of 185 miRNAs were detected in at least 10% of samples. The detectable rate varied for each miRNA, with 69 and 44 miRNAs being detected in 50% and 75% of samples, respectively. The median ICC was 0.46 for all of the 185 detectable miRNAs. Among them, 41% (75 miRNAs) had an ICC ≥0.5, and 42 miRNAs had an ICC ≥0.6. The ICC is higher for miRNAs with higher expression levels and higher detection rates, when compared to their counterparts with lower expression or detection rates. In summary, we found that 185 miRNAs were expressed in at least 10% of plasma samples, and 69 miRNAs in 50% of samples from healthy Chinese men and women, and that 75 of these miRNAs had an ICC ≥0.5. These results suggest that common circulating miRNAs can serve as reliable biomarkers for epidemiological and clinical research.

Citation Format: Jie Wu, Hui Cai, Yong-Bing Xiang, Charles Matthews, Fei Ye, Wei Zheng, Qiuyin Cai, Xiao-Ou Shu. Intra-individual variation in circulating miRNA expression levels in human plasma samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4285.
Abstract 4288: Association between tumor necrosis factor alpha -308 G/A polymorphism and risk of bladder cancer in Asian population: a meta-analysis
Authors: Kao Wei-Tang , Tung Min-Che , Hsueh Kuan-Chun , Wang Yuan-Hung , Wu Chia-Chang
Published: July 2016

Previous studies have investigated the association between TNF-α-308 G/A polymorphism and bladder cancer risk in various populations. However, these findings remain inconclusive. Therefore, we performed a meta-analysis to explore the relationship between TNF-α-308 G/A polymorphism and bladder cancer risk. A literature search in PubMed was performed to select eligible studies regarding the association between TNF-α-308 G/A polymorphism and bladder cancer risk. The strength of risk under fixed- and random- effects models were estimated using the odds ratios (ORs) with 95% confidence intervals (CIs). We identified 7 case-control studies including 1153 cases and 1587 controls were included in the present meta-analysis. Compared with subjects carrying the G/G genotype of TNF-α-308 G/A polymorphism, those with the A/A and G/A genotypes had non-significant bladder cancer risks under the fixed effects model (OR = 1.037) and the random effects model (OR = 1.012). In the recessive model, subjects with the A/A genotype had an increased bladder cancer risk (OR = 1.875) compared with those carrying the G/A and G/G genotypes of TNF-α-308 G/A polymorphism. The major finding of this meta-analysis suggest that TNF-α-308 G/A polymorphism is correlated with the risk of bladder cancer in Asian population under the recessive model. We should further investigate the joint effects of environmental risk factors and TNF-α-308 G/A polymorphism on bladder cancer risk.

Citation Format: Wei-Tang Kao, Min-Che Tung, Kuan-Chun Hsueh, Yuan-Hung Wang, Chia-Chang Wu. Association between tumor necrosis factor alpha -308 G/A polymorphism and risk of bladder cancer in Asian population: a meta-analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4288.
Abstract 4292: Mitochodrial D-loop polymorphisms are associated with colorectal adenoma risk
Authors: Thyagarajan Bharat , Guan Weihua , Fedirko Veronika , Barcelo Helene , Gross Myron D. , Goodman Michael , Bostick Roberd M.
Published: July 2016

Somatic mutational events in the mitochondria have been detected in adenomatous polyps. However, the contribution of germline variation in the mitochondrial DNA towards risk of colorectal adenomas, well recognized precursor lesions to colorectal cancer, has not been evaluated previously. Hence, we evaluated associations of mitochondrial polymorphisms in the D-loop and non-D loop regions with incident colorectal adenoma in three pooled colonoscopy-based case-control studies (n = 327 colorectal adenoma cases and 420 controls) that used identical methods for case ascertainment and risk factor determination. We sequenced a 1,124 bp fragment to comprehensively identify all genetic variation in the mitochondrial D-loop region, and used the Sequenom platform to genotype 64 previously described tagSNPs in the non-D loop region. We used multivariate unconditional logistic regression to analyze the association between the mitochondrial polymorphisms and colorectal adenoma risk after adjustment for potential confounders. We identified 320 germline mutations in the D-loop region; 30 (9%) had a minor allele frequency (MAF) ≥ 5%. Most of the mutations clustered in the hypervariable regions HV1 (n = 124; 39%) and HV2 (n = 111; 35%) of the D-loop region. Among the nine common polymorphisms (MAF ≥ 5%) in the HV1 region, four polymorphisms (mt16069, mt16278, mt16294, and mt16296) were statistically significantly directly associated with colorectal adenoma risk (odds ratios [OR]: 1.76 - 2.66; p-values: 0.001 - 0.04). In addition, the polyC tract in the HV1 region was inversely associated with colorectal adenoma risk (OR: 0.90; p = 0.03). None of the other polymorphisms in the mitochondrial D-loop region tract was associated with colorectal adenoma risk. After correction for multiple comparisons, none of the mitochondrial tagSNPs in the non-D loop region was associated with colorectal adenoma risk. These findings suggest that polymorphisms in the HV1 region of the mitochondrial D-loop may be associated with colorectal adenoma risk and support further investigation in future studies.

Citation Format: Bharat Thyagarajan, Weihua Guan, Veronika Fedirko, Helene Barcelo, Myron D. Gross, Michael Goodman, Roberd M. Bostick. Mitochodrial D-loop polymorphisms are associated with colorectal adenoma risk. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4292.
Abstract 4299: Short telomeres are associated with increased risk for arsenic-related skin lesions in Bangladesh
Authors: Zhang Chenan , Kibriya Muhammad , Jasmine Farzana , Roy Shantanu , Ahsan Habibul , Pierce Brandon L.
Published: July 2016

Chronic exposure to arsenic, a human carcinogen, has been shown in epidemiologic studies to increase the risk of arsenical skin lesions (keratosis and melanosis) as well as cancer (including non-melanoma skin cancer). Recent studies suggest that the mechanism of arsenic's toxicity may be related to telomere dysfunction, including altered telomere length (TL), and differential expression of genes involved in telomere maintenance. However, prior studies of arsenic exposure, TL, and skin lesion tend to be small and cross-sectional in nature. In this study, we investigated the association between arsenic exposure and TL (measured by qPCR as T/S ratio), as well as TL and subsequent risk of skin lesion in individuals who are exposed to naturally contaminated drinking water with a wide range of arsenic levels in the Health Effects of Arsenic Longitudinal Study (HEALS) in Araihazar, Bangladesh (2000-2009). In all laboratory analyses, we attempted to randomize samples across plate positions, and in statistical analyses, we controlled for any residual confounding by plate or plate position. All TL data generated showed a strong inverse association with age. In a random sample of the baseline cohort (n = 649), we observed a positive association between water arsenic levels and TL (Ptrend = 0.035), with beta estimates of 0.001, 0.023, and 0.025 for water arsenic levels of 10.1-50ug/L, 50.1-150ug/L and 150+ug/L, respectively, compared to <10ug/L. However the association was not replicated in an independent random sample (n = 1181, Ptrend = 0.176) with beta estimates of 0.007, -0.004, and -0.017. In a nested case-control study comparing skin lesion incident cases diagnosed across a nine year period with sex- and age-matched controls (448 cases and 462 controls), we observed higher incident skin lesion risk with shorter TL (Ptrend = 4.6E-5), with odds ratios of 3.05, 1.30, and 1.21 for the first (shortest), second, and third TL quartiles compared to the longest. Our findings show that arsenic exposure is not strongly associated with TL, indicating that TL is likely not the main mode of action for arsenic's carcinogenic toxicity. However, TL is inversely associated with skin lesion risk, suggesting that short TL may be a biomarker of susceptibility for arsenical skin lesions independent of arsenic exposure.

Citation Format: Chenan Zhang, Muhammad Kibriya, Farzana Jasmine, Shantanu Roy, Habibul Ahsan, Brandon L. Pierce. Short telomeres are associated with increased risk for arsenic-related skin lesions in Bangladesh. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4299.
Abstract 4309: Rare protein truncating and missense variants in ATM, CHEK2, PALB2, but not XRCC2, confer increased breast cancer risks
Authors: Decker Brennan , Allen Jamie , Luccarini Craig , Pooley Karen A. , Pharoah Paul PD , Dunning Alison M. , Easton Douglas F.
Published: July 2016

Breast cancer (BC) is the most common malignancy in women and has a major heritable component that is driven by both common SNPs and rarer variants in susceptibility genes. However, the risks associated with most rare susceptibility alleles are not well estimated. We sequenced the coding regions of ATM, CHEK2, PALB2, and XRCC2 in 13,087 BC cases and 5,488 controls from East Anglia to characterize their role in BC risk in the largest sequencing study of these genes to date.

We performed multiplexed microfluidic PCR target enrichment of 221 amplicons, covering 99.3% of the coding bases. Amplified DNA was barcoded, pooled, and sequenced on the Illumina HiSeq2000. Reads were demultiplexed, trimmed, and aligned to the hg19 reference sequence. Variants were called with GATK UnifiedGenotyper and filtered for missing data. Retained variants were annotated with CADD and VEP. Truncating variants, missense alleles, and other functional variant groups were assessed for association with BC risk using logistic regression.

Truncating variants in ATM, CHEK2, and PALB2 increased overall BC risk, whereas XRCC2 variants did not (Table 1). The estimated relative risk was higher for PALB2 than ATM or CHEK2. While 88% of CHEK2 variant alleles were attributable to CHEK2*1100delC, other truncating alleles appeared to confer similar risks. CHEK2 truncating alleles were more strongly associated with risk of estrogen receptor (ER) positive disease than ER negative or triple negative disease. In contrast, PALB2 protein-truncating variants conferred similar relative risks for all subtypes. Analysis of rare missense variants suggested that specific subset of alleles in ATM (P = 0.012), CHEK2 (P = 0.009), and PALB2 (P = 0.017) may contribute to risk. Localization within known functional domains were a predictor of association among these variants.

These results confirm that truncating variants in ATM, CHEK2, and PALB2 are associated with moderate BC risks, and exclude a large effect on risk from XRCC2 variants. Truncating Variant RiskATMATMCHEK2CHEK2PALB2PALB2XRCC2XRCC2ORP-ValueORP-ValueORP-ValueORP-ValueSEARCH Cases3.262.11E-053.345.18E-115.424.45E-080.940.9232ER Positive3.191.04E-043.719.11E-124.322.73E-050.870.8419ER Negative1.280.67431.560.23655.583.62E-040.880.9102Triple Negative2.850.14880.420.32217.500.0022NANA

Citation Format: Brennan Decker, Jamie Allen, Craig Luccarini, Karen A. Pooley, Paul PD Pharoah, Alison M. Dunning, Douglas F. Easton. Rare protein truncating and missense variants in ATM, CHEK2, PALB2, but not XRCC2, confer increased breast cancer risks. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4309.
Abstract 4318: Reversal of BRCA-1 CpG hyperthylation by genistein and (-)-epigallocatechin-3-gallate in human breast cancer cells with activated AhR
Authors: Selmin Ornella I. , Papoutsis Andreas J. , Romagnolo Donato F.
Published: July 2016

Sporadic breast cancers, which represent the vast majority (∼90%) of breast tumor cases, do not have mutations in the tumor suppressor gene, BRCA-1 but have absent or markedly reduced levels of BRCA-1 similar to those observed in hereditary BRCA-1 tumors. Therefore, understanding the non-mutational mechanisms that contribute to repression of BRCA-1 has important implications for the prevention of both hereditary and sporadic breast tumors. Agonists of the aromatic hydrocarbon receptor (AhR) are ubiquitous in the environment and include dietary compounds, metabolites of fatty acids, industrial pollution, and photoproducts generated in the skin from ultraviolet radiation. In cultured estrogen-receptor (ER)α-positive MCF-7 breast cancer cells, we found that activation of the AhR with the prototype ligand 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) repressed by 50% estradiol-induced BRCA-1 expression. Conversely, the post-treatment with the soy isoflavone genistein and tea (-)-epigallocatechin-3-gallate (EGCG) at physiological doses (1 μM) rescued within 24 to 48 h BRCA-1 expression to levels ∼2.0 to 3.0-fold higher than those measured in cells treated with estradiol alone. Genistein and EGCG reversed BRCA-1 promoter hypermethylation in AhR-activated MCF-7 cells. In ERα-negative and AhR-overexpressing UACC-3199 breast cancer cells, genistein (1 μM) reactivated BRCA-1 expression. The post-treatment of AhR-activated MCF-7 cells with genistein and EGCG antagonized the recruitment of DNA-methyl transferase-1 (DNMT-1) enzyme to the BRCA-1 promoter. These results suggest the involvement of epigenetic mechanisms in the repression of BRCA-1 and reversal effects of genistein and EGCG against breast tumorigenesis associated with activation of the AhR.

Citation Format: Ornella I. Selmin, Andreas J. Papoutsis, Donato F. Romagnolo. Reversal of BRCA-1 CpG hyperthylation by genistein and (-)-epigallocatechin-3-gallate in human breast cancer cells with activated AhR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4318.
Abstract 4332: Discovery of BAY 1163877 - A pan-FGFR inhibitor: De novo structure-based design and lead optimization of benzothiophenyl-pyrrolotriazines
Authors: Collin Marie-Pierre L. , Lobell Mario , Huebsch Walter , Brohm Dirk , Héroult Mélanie , Lustig Klemens , Gruenewald Sylvia , Boemer Ulf , Jautelat Rolf , Hess-Stump Holger , Jaroch Stefan , Brands Michael , Ziegelbauer Karl
Published: July 2016

Fibroblast growth factors (FGFs) orchestrate a variety of cellular functions by binding to their transmembrane tyrosine-kinase receptors (FGFR1-4) and activating downstream signaling pathways. Alterations in FGFR encoding genes are frequently observed in a variety of solid tumors including lung, gastric, breast and urothelial cancer. Therefore, targeting FGFRs using selective FGFR inhibitors is an attractive therapeutic approach to treat cancer patients.

BAY 1163877 is an orally active, highly potent and selective small molecule FGFR-1, -2 and -3 kinase inhibitor. We disclose for the very first time its discovery and chemical structure. BAY 1163877 was derived from a de novo structure-based design approach and medicinal chemistry optimization. Data on the structure activity relationship and the pharmacokinetic profile of the benzothiophenyl-pyrrolotriazine structure class will be presented. Based on its favorable preclinical profile, BAY 1163877 is currently being investigated in a Phase 1 clinical trial (NCT01976741).

Citation Format: Marie-Pierre L. Collin, Mario Lobell, Walter Huebsch, Dirk Brohm, Mélanie Héroult, Klemens Lustig, Sylvia Gruenewald, Ulf Boemer, Rolf Jautelat, Holger Hess-Stump, Stefan Jaroch, Michael Brands, Karl Ziegelbauer. Discovery of BAY 1163877 - A pan-FGFR inhibitor: De novo structure-based design and lead optimization of benzothiophenyl-pyrrolotriazines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4332.
Abstract 4351: A neighborhood-wide association study (NWAS) in prostate cancer: A new methodologic approach
Authors: Lynch Shannon , Mitra Nandita , Ross Michelle , Newcomb Craig , Daily Karl , Jackson Tara , Zeigler-Johnson Charnita , Riethman Harold , Branas Charles , Rebbeck Timothy
Published: July 2016

Background: Cancer results from complex interactions of multiple variables at biological, individual and social levels. However, comprehensive, empirical methods to assess social and neighborhood-level effects are limited. We propose a Neighborhood-Wide Association Study(NWAS), analogous to a genome-wide association study(GWAS), to comprehensively identify neighborhood signatures associated with disease. Methods: Pennsylvania State Cancer Registry data from 1995-2005 were linked to the 2000 U.S. Census data. In a successively more stringent multiphase approach, we evaluated the association between neighborhood exposures(n = 14,663, standard deviation adjusted census variables) and prostate cancer aggressiveness (n = 6,416 aggressive Stage>3/Gleason grade >7 cases vs. n = 70,670 non-aggressive Stage<3/Gleason grade<7 controls) in White men. Each phase accounted for age, year of diagnosis, spatial correlation, and multiple testing. We used generalized estimating equations in Phase 1 and Bayesian mixed effects models in Phase 2 to calculate odds ratios(OR) and credible intervals(CI). Variables meeting statistically significant thresholds after Bonferroni adjustment were identified and used in subsequent phases. In Phase 3, principal components analysis grouped correlated variables.

Results: From 14,663 census variables, 17 variables that were most significantly associated with prostate cancer aggressiveness were identified. The top two hits related to transportation (OR = 1.05;CI = 1.001-1.09) and poverty (OR = 1.07;CI = 1.01-1.12). Significant associations between prostate cancer and income, housing, employment, and immigration were also found.

Conclusions: This NWAS methodology addresses gaps in neighborhood and cancer research by proposing a standardized, agnostic evaluation of neighborhood using readily accessible data from the U.S. Census. This approach has implications for health disparities research and can be applied broadly to large-scale, public health data.

Citation Format: Shannon Lynch, Nandita Mitra, Michelle Ross, Craig Newcomb, Karl Daily, Tara Jackson, Charnita Zeigler-Johnson, Harold Riethman, Charles Branas, Timothy Rebbeck. A neighborhood-wide association study (NWAS) in prostate cancer: A new methodologic approach. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4351.
Abstract 4357: Harnessing system xCT- to target mutant p53 cancer cells
Authors: Liu David SH , Read Matthew , Wiman Klas G. , Haupt Sue , Duong Cuong P. , Abrahmsen Lars , Haupt Ygal , Clemons Nicholas J. , Phillips Wayne A.
Published: July 2016

p53, a critical tumour suppressor is mutated in over half of all human cancers. The loss of wild-type p53 activity together with oncogenic gain-of-function, secondary to aberrant accumulation of mutant p53 protein frequently results in aggressive tumour phenotype, resistance to conventional therapies and poor survival. Therefore, effective therapeutic strategies to target mutant p53 cancer cells remain an urgent and unmet medical need. Here we show that mutant p53 accumulation across multiple tumour types represses the transcription of SLC7A11, a key component of system xCT-, resulting in reduced cystine uptake, lowering endogenous glutathione stores and predisposing cells to oxidative damage. Notably, genetic knockdown or pharmacological inhibition (erastin and sulfasalazine) of system xCT- preferentially induces apoptosis in cancer cells with mutant p53 accumulation. Moreover, we found that APR-246 (PRIMA-1met), a first-in-class reactivator of mutant p53 currently in early clinical trials, depletes cellular glutathione and induces significantly higher amounts of reactive oxygen species in mutant p53 cancer cells compared with normal cells. This leads to lipid peroxidation of mitochondrial membranes and the release of matrix contents, culminating in apoptotic cell death. Conversely, APR-246-induced cytotoxicity could be rescued by cysteine or glutathione replacement, or with lipophilic antioxidants. In extension, we identified and functionally validated SLC7A11 expression as a specific predictive biomarker for APR-246. Importantly, we demonstrate that antagonising system xCT- activity in combination with APR-246 selectively and synergistically inhibit mutant p53 cancer cells. Together, our findings propose that accumulation of mutant p53 protein in cancer cells, through its repressive effects on SLC7A11 expression, creates an ‘Achilles heel’ that can be targeted by further perturbations of the glutathione pathway.

Citation Format: David SH Liu, Matthew Read, Klas G. Wiman, Sue Haupt, Cuong P. Duong, Lars Abrahmsen, Ygal Haupt, Nicholas J. Clemons, Wayne A. Phillips. Harnessing system xCT- to target mutant p53 cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4357.
Abstract 4359: Rescue of exhausted CD8 T cells by PD-1 targeted therapies is CD28-dependent
Authors: Kamphorst Alice O. , Wieland Andreas , Yang Shu , Nasti Tahseen , Zhang Ruan , Barber Daniel L. , Konieczny Bogumila T. , Koenig Lydia , Yu Ke , Sica Gabriel , Owonikoko Taofeek K. , Pillai Rathi , Ramalingam Suresh S. , Sharpe Arlene H. , Freeman Gordon J. , Turka Laurence A. , Araki Koichi , Ahmed Rafi
Published: July 2016

T cell exhaustion was first described in mice during chronic lymphocytic choriomeningitis virus (LCMV) infection and later shown to occur in humans during persistent infections and cancer. Sustained expression of the inhibitory receptor programmed cell death-1 (PD-1) is a hallmark of T cell exhaustion. Therapeutic blockade of PD-1 rescues proliferation, cytokine production and cytotoxicity of exhausted T cells. PD-1 targeted therapies have shown efficacy for multiple cancer types and changed the landscape of cancer treatment, but the requirements for T cell rescue by blockade of the PD-1 pathway remain elusive. T cell activation ensues when positive signals overcome negative signals. CD28 is the most prominent positive co-receptor for T cell activation, and human CD8 T cells become CD28neg upon continuous stimulation. We analyzed tumor specimens from non-small cell lung cancer (NSCLC) patients by flow cytometry and found heterogeneous CD28 expression on tumor-infiltrating CD8 T cells. In most NSCLC patients, CD28neg cells were prevalent among CD8 T cells co-expressing inhibitory receptors (such as PD-1 and Tim-3). To address the role of the CD28/B7 pathway on CD8 T cell rescue by PD-1 targeted therapies, we used the LCMV chronic infection model. When B7 signals were impeded by CTLA-4-Ig or B7 blocking antibodies, blockade of the PD-1 pathway failed to rescue virus-specific CD8 T cells. To investigate a cell intrinsic requirement of CD28 on exhausted CD8 T cells, we used an adoptive transfer strategy of cells genetically deficient in CD28. We found that CD28-deficient LCMV-specific CD8 T cells do not expand after blockade of the PD-1 pathway. Furthermore, to ensure appropriate development and differentiation of CD8 T cells, we used mice in which the CD28 gene can be conditionally deleted. We confirmed that CD28neg exhausted CD8 T cells failed to proliferate after blockade of the PD-1 pathway even when CD28 gene deletion was induced on already exhausted T cells. These data demonstrate that CD28 engagement is required for CD8 T cell rescue, and CD28 status may constitute a predictive biomarker for PD-1 targeted therapies.

Citation Format: Alice O. Kamphorst, Andreas Wieland, Shu Yang, Tahseen Nasti, Ruan Zhang, Daniel L. Barber, Bogumila T. Konieczny, Lydia Koenig, Ke Yu, Gabriel Sica, Taofeek K. Owonikoko, Rathi Pillai, Suresh S. Ramalingam, Arlene H. Sharpe, Gordon J. Freeman, Laurence A. Turka, Koichi Araki, Rafi Ahmed. Rescue of exhausted CD8 T cells by PD-1 targeted therapies is CD28-dependent. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4359.
Abstract 4370: Genome-wide CRISPR-Cas9 screens reveal loss of redundancy between PKMYT1 and WEE1 in patient-derived glioblastoma stem-like cells
Authors: Toledo Chad , Yu Ding , Hoellerbauer Pia , Davis Ryan , Basom Ryan , Girard Emily , Corrin Philip , Bolouri Hamid , Davison Jerry , Zhang Qing , Nam Do-Hyun , Lee Jeongwu , Pollard Steven , Delrow Jeffery , Clurman Bruce , Olson James , Paddison Patrick
Published: July 2016

Glioblastoma multiforme (GBM) is the most aggressive and common form of brain cancer in adults. There are currently no effective therapies for GBM. Even with standard of care treatments, such as surgery, radiation, and chemotherapy, ∼90% of adult patients die within 2 years of diagnosis. To identify therapeutic targets for Glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 “knockout” (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit Cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, likely as a result of oncogenic signaling, causing GBM-specific lethality. From a biological standpoint, our results help re-discover PKMYT1 function in human cells. From a cancer standpoint, these results suggest that targeting PKMYT1 is a glioma-lethal gene and that tandem or sequential use of PKMYT1 and WEE1 inhibitors may illicit therapeutic responses in GBM. In addition to these results we will also present retest data for other candidate glioma-lethal genes and networks in multiple patient samples.

Citation Format: Chad Toledo, Ding Yu, Pia Hoellerbauer, Ryan Davis, Ryan Basom, Emily Girard, Philip Corrin, Hamid Bolouri, Jerry Davison, Qing Zhang, Do-Hyun Nam, Jeongwu Lee, Steven Pollard, Jeffery Delrow, Bruce Clurman, James Olson, Patrick Paddison. Genome-wide CRISPR-Cas9 screens reveal loss of redundancy between PKMYT1 and WEE1 in patient-derived glioblastoma stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4370.
Abstract 4386: CETSA as a new strategy to understand efficacy, adverse effects and resistance development of anticancer drugs
Authors: Nordlund Pär , Lööf Sara , Laursen Henritte , Öberg Anette , Lengqvist Johan , Jafari Rozbeh , Dai Lingyun , Go Ka DIam , Prabhu Nayana , Sobota Radoslaw , Larsson Andreas , Jansson Anna , Soon Chris Heng Tan , Sreekumar Lekshmy , Lim Yan Ting , Martines Molina Daniel
Published: July 2016

A key step of the action of most drugs is their binding (engagement) of the target protein(s). However, limitations in the available methods for directly accessing this critical step have added uncertainties in many stages of drug development.

We have developed a generic method for evaluating drug binding to target proteins in cells and tissues (Martinez Molina et al. Science, 341:84). The technique is based on the physical phenomenon of ligand-induced thermal stabilization of target proteins; the method is therefore called the cellular thermal shift assay (CETSA). The technique allows for the first time to directly measure the biophysical interactions between a drug and protein target in non- engineered cells and tissues. We show that using CETSA a range of critical factors for drug development can be addressed at the target engagement level, including drug transport and activation, off-target effects, drug resistance as well as drug distribution in cells, patient and animal tissues. Using quantitative mass-spectrometry, proteome-wide CETSA has been established which allows for off-target effects as well as downstream biochemistry to be discovered (Savitsk et al. Science, 346, 6205:1255784). Together the data supports that CETSA is likely to become a valuable tool for developing and understanding the action of cancer drugs in the future.

Citation Format: Pär Nordlund, Sara Lööf, Henritte Laursen, Anette Öberg, Johan Lengqvist, Rozbeh Jafari, Lingyun Dai, Ka DIam Go, Nayana Prabhu, Radoslaw Sobota, Andreas Larsson, Anna Jansson, Chris Heng Tan Soon, Lekshmy Sreekumar, Yan Ting Lim, Daniel Martines Molina. CETSA as a new strategy to understand efficacy, adverse effects and resistance development of anticancer drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4386.
Abstract 4389: Therapeutic opportunities of RANK pathway in breast cancer
Authors: Perez Montoyo Hector , Gomez Miragaya Jorge , Trinidad Eva M. , Martinez-Aranda Antonio , Soler Monso Maria Teresa , Petit Anna , Sierra Angels , Gonzalez Suarez Eva
Published: July 2016

RANK pathway, key in bone remodeling and metastasis, is essential for mammary gland development. Using genetic mouse models we have previously shown that RANKL is the main mediator of the protumorigenic role of progesterone in the mouse mammary gland. RANK overexpression in non-transformed human mammary cell lines induces epithelial to mesenchymal transition and stemness, and promotes tumorigenesis and metastasis in breast cancer lines. RANK is expressed in a subset of basal and luminal human adenocarcinomas.

Our goal is to determine a putative association of RANK and RANKL expression in breast adenocarcinomas with clinical parameters and to evaluate the functional role of RANK signaling using patient derived orthoxenografts of breast cancer. We have analyzed RANK and RANKL expression levels in an extensive collection of human breast adenocarcinomas collected from multiple centers, enriched in metastatic tumor samples. From the 318 patients with breast adenocarcinomas 138 developed metastasis. RANK expression is found on tumor cells but also on stromal cells, including tumor associated macrophages. RANKL is strongly expressed in some tumors, although the frequency is low. Analyses of RANK and RANKL mRNA and protein expression has been performed in multiple models of patient derived breast orthoxenografts. Several models with variable levels of RANK as well as RANKL expression have been identified and established in the laboratory. Functionality of the pathway was corroborated analyzing NF-kB activation upon in vitro and in vivo treatments with RANKL, in order to activate RANK signaling, or with RANK-Fc to inhibit it. We found that RANKL treatment induced NF-kB activation even in tumor cells with low levels of RANK expression.

Citation Format: Hector Perez Montoyo, Jorge Gomez Miragaya, Eva M. Trinidad, Antonio Martinez-Aranda, Maria Teresa Soler Monso, Anna Petit, Angels Sierra, Eva Gonzalez Suarez. Therapeutic opportunities of RANK pathway in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4389.
Abstract 4400: Hematopoietic age at onset of breast cancer dictates disease aggressiveness and progression
Authors: Sceneay Jaclyn , Marsh Timothy , Wong Irene , Qin Yuanbo , Sjödin Andreas , Nilsson Björn , Stewart Sheila A. , McAllister Sandra S.
Published: July 2016

Aging is strongly associated with increased risk of developing breast cancer. Young age at diagnosis is typically associated with more aggressive disease, while breast cancer is generally more indolent in older women. Among the different breast cancer subtypes, triple negative breast cancer (TNBC) accounts for ∼15% of invasive breast cancer cases and TNBC patients have a poor prognosis due to lack of targeted therapies and limited sensitivity to chemotherapy. Emerging studies indicate that older women with TNBC have a better outcome than younger women for reasons that are unclear. Nevertheless, older patients tend to suffer co-morbidities associated with chemotherapy and often forego treatment, resulting in disease recurrence and a poor outcome for these patients. Treatment of TNBC therefore presents age-specific challenges.

Here, we investigated how age affects the composition and function of pro-tumorigenic bone marrow cells using TNBC models that enable us to interrogate the impact of bone marrow derived cells on both tumor cells and the tumor microenvironment. Consistent with clinical observations, we found that disease progression was significantly more aggressive in young mice relative to aged mice. We identified age- and tumor-dependent molecular and functional changes to bone marrow derived hematopoietic cells that impact TNBC progression. A specific population of Sca1+/cKit- bone marrow cells that expresses CSF1R and secretes the growth factor granulin to support stromal activation and robust tumor growth in young mice, fails to promote tumor outgrowth in aged mice, resulting in maintenance of disease indolence. Importantly, bone marrow cells from young mice are sufficient to activate a tumor-supportive microenvironment and rescue tumor progression in aged mice.

The results presented here indicate that hematopoietic age is an important determinant of TNBC aggressiveness and provide rationale for investigating age-stratified therapies designed to prevent activation of tumor-promoting bone marrow cells.

Citation Format: Jaclyn Sceneay, Timothy Marsh, Irene Wong, Yuanbo Qin, Andreas Sjödin, Björn Nilsson, Sheila A. Stewart, Sandra S. McAllister. Hematopoietic age at onset of breast cancer dictates disease aggressiveness and progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4400.
Abstract 4405: Over-expression of PARP is associated with an aggressive phenotype and can be synergistically targeted using combination of PARP and XIAP inhibitors in breast cancer
Authors: Ahmed Maqbool , Hussain Azhar R. , Beg Shaham , Thangavel Saravanan , Begum Rafia , Ajarim Dahish , Al-Dayel Fouad , Siraj Abdul Khalid Siraj K. , Al-kuraya Khawla S.
Published: July 2016

Breast cancer is the most common cancer in females and despite improvement in treatment modality with the introduction of hormonal targeted therapy, these cancers still remains a major cause of morbidity and mortality in females. Poly ADP ribose polymerase (PARP) is an enzyme which plays a critical role for repairing single-strand breaks. The main function of PARP1 is dependent upon cellular stress responses and directs cells towards DNA repair based on the strength of the stress stimulus. PARP has been found to be over-expressed in various tumors including ovarian cancer; breast cancer and lung cancer, however, its role in the pathogenesis of breast cancer of Middle Eastern origin have not been completely elucidated. Therefore, we examined the expression of PARP in a large cohort of more than 1000 breast cancer by IHC in a tissue microarray format and found PARP was over expressed in 44.7% (451/1008) and was found to be significantly associated with aggressive markers such as Triple negative phenotype, high grade tumors, large tumor size, stage 4 disease and had a poor 5 year overall survival of 72.6 months as compared to 85.4 months (p = 0.0005). Interestingly PARP expression was also found to be significantly associated with aggressive molecular markers such as activated AKT, XIAP and proliferative marker; ki67. Next, in vitro effect of inactivation of PARP was evaluated using a PARP inhibitor; olaparib on a panel of BC cell lines. Olaparib treatment alone did not affect the cell viability and apoptosis at doses up to 10μM concentration. However, when BC cells were treated with sub-optimal dose of Olaparib (1μM) and Embelin (XIAP inhibitor)(5μM) in combination for 48 hours, there was appreciable inhibition of cell viability via induction of apoptosis. Combination of Olaparib and Embelin induced apoptosis via activation and cleavage of caspases-9, -3 and PARP in BC cells. These studies highlight the importance of targeting PARP in combination with either small molecular inhibitors or chemotherapeutic agents in BC can improve the management and survival of this aggressive disease.

Citation Format: Maqbool Ahmed, Azhar R. Hussain, Shaham Beg, Saravanan Thangavel, Rafia Begum, Dahish Ajarim, Fouad Al-Dayel, Abdul Khalid Siraj K. Siraj, Khawla S. Al-kuraya. Over-expression of PARP is associated with an aggressive phenotype and can be synergistically targeted using combination of PARP and XIAP inhibitors in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4405.
Abstract 4406: Inducible knockdown of insulin receptor substrate I desensitizes ER alpha response to both agonist (estradiol) and antagonist (fulvestrant) in MCF-7L breast cancer cells
Authors: Zhang Xihong , Varma Sidhant , Yee Douglas
Published: July 2016

Insulin receptor substrate (IRS) proteins are adaptor proteins phosphorylated by insulin and insulin-like growth factor (IGF) receptors. In addition to their roles in regulating IGF and insulin responses, IRS proteins function in other pathways in normal and malignant physiology. Our data suggested that IRS1 enhanced IGF/insulin stimulated proliferation while IRS-2 was important in regulating motility. Therefore IRS proteins could be potential therapeutic target for cancer therapy. We have previously shown that reduced IRS1 can hinder cancer cell growth and tumorigenicity stimulated by IGF-I, IGF-II and insulin. In this study, we evaluated whether inducible IRS1 suppression affected estrogen receptor-alpha (ER) responsiveness to ER agonist estradiol and antagonist fulvestrant. We evaluated several doxycycline inducible shRNA IRS1 constructs (Thermo Scientific pTRIPZ inducible Lentiviral IRS1 shRNA) expressed in the ER+ MCF-7L cell line. Doxcycline treatment reduced IRS1 mRNA and protein levels confirmed by qRT-PCR and immunoblot in several independently selected clones. We used a representative clone (3G5) to evaluate effects on ER function. After IRS1 inducible knockdown, estradiol (E2) stimulated growth was suppressed. To investigate if reduced IRS1 regulated E2 stimulated binding of ER to the pS2 estrogen response element (ERE), chromatin immunoprecipitation (ChIP) assays were performed. Compared to non-induced MCF-7L cells, inducible IRS1 suppression demonstrated significantly reduced binding of ER to this promoter. The pharmacologic antagonist of IRS proteins, NT157, also resulted in diminished ER binding to pS2 promoter. mRNA of several ER regulated genes, such as PGR, TFF1, etc, showed downregulation in response to E2 when IRS-1 was suppressed. We have shown that the pure steroidal ER antagonist fulvestrant activates EGFR signaling in an ER dependent manner through upregulation of EGFR ligands. However, inducible IRS1 suppression blocked this ER-dependent activation of EGFR phosphorylation by fulvestrant yet EGFR phosphorylation remained intact when stimulated by addition of EGF. These data suggest that regulation of EGFR ligands by fulvestrant also requires IRS-1 expression. We conclude that ER function is affected by IRS-1 expression. Thus, IRS-1 may also be a target in endocrine responsive and resistant breast cancers.

Citation Format: Xihong Zhang, Sidhant Varma, Douglas Yee. Inducible knockdown of insulin receptor substrate I desensitizes ER alpha response to both agonist (estradiol) and antagonist (fulvestrant) in MCF-7L breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4406.
Abstract 4410: ZEB2 drives cell motility and metastasis in ER+ breast cancer cells through a novel, E-cadherin independent pathway
Authors: Burks Hope E. , Rhodes Lyndsay V. , Martin Elizabeth C. , Hoang Van T. , Elliott Steven , Badoo Melody , Phamduy Theresa , Buechlein Aaron , Rusch Douglas , Chrisey Douglas , Flemington Erik , Nephew Kenneth , Collins-Burow Bridgette , Burow Matthew E.
Published: July 2016

Breast cancer is the most commonly diagnosed cancer in women, with the second highest mortality rate. The overwhelming majority of breast cancer deaths are a direct result of distant metastatic spread to vital organs such as lung, bone, and brain. The zinc finger transcription factor ZEB2 has been implicated as a driver of cancer cell motility, tissue invasion and metastasis in a number of diverse malignancies, but its role in breast cancer is not completely understood. We chose to examine the effects of direct overexpression of ZEB2 in the MCF-7 cell line, a luminal and estrogen receptor positive (ER+) derived breast cancer line. MCF-7-ZEB2 cells demonstrated significantly increased migration and invasion as well as an altered morphology in vitro compared to that of vector. Additionally, MCF-7-ZEB2 cells exhibited increased lung metastasis in vivo when implanted in an orthotopic xenograft mouse model. ZEB2 function in metastasis has canonically been attributed to transcriptional repression of the cell junction protein E-Cadherin. Here we establish that in ER+ breast cancer cells, ZEB2 fails to repress E-cadherin and promotes cell motility and metastasis through the induction of an E-cadherin independent signaling cascade. Next generation RNA sequencing analysis of the MCF-7-ZEB2 cells compared to vector revealed alteration of the MAPK signaling cascade, evidenced by enhanced expression of key motility and MAPK associated genes, including PLAU, EGF, ACTA2, and MMP9. Pharmacological inhibition of MAPK pathway completely abrogated ZEB2 induced migration, cell morphology changes and expression of target motility genes, confirming a necessary role for MAPK signaling in ZEB2-driven cell motility in ER+ breast cancer. Together these results indicate that the ZEB2 transcription factor drives motility in breast cancer cells through a novel MAPK dependent pathway, warranting further investigation into the mechanisms involved in ZEB2 action. Elucidating the pathways involved in ZEB2 function which are specific to ER+ breast cancer is an important step in understanding the processes underlying metastasis and has the potential to yield new therapeutic targets.

Citation Format: Hope E. Burks, Lyndsay V. Rhodes, Elizabeth C. Martin, Van T. Hoang, Steven Elliott, Melody Badoo, Theresa Phamduy, Aaron Buechlein, Douglas Rusch, Douglas Chrisey, Erik Flemington, Kenneth Nephew, Bridgette Collins-Burow, Matthew E. Burow. ZEB2 drives cell motility and metastasis in ER+ breast cancer cells through a novel, E-cadherin independent pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4410.
Abstract 4412: c-Myc-driven lymphomas require calcium-modulating cyclophilin ligand (CAML) for survival and proliferation
Authors: Shing Jennifer C. , Lindquist Lonn D. , Bram Richard J.
Published: July 2016

Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum (ER) protein that functions in a complex to mediate tail-anchored (TA) protein insertion into the ER membrane. CAML has also been implicated as a regulator of endocytic trafficking, modulator of intracellular calcium signaling, and essential factor for the survival and proliferation of specialized immune cells. To investigate the role of CAML in cell biology and cancer, we generated Eμ-Myc transgenic mice that carry a conditional deletion allele of CAML. From these mice, we derived B cell lymphoma cell lines driven by c-Myc and induced deletion of the CAML gene by treatment with tamoxifen to activate Cre-ERT2 in vitro. Homozygous loss of CAML in multiple independent B cell lymphoma lines activated apoptosis, while heterozygous deletion led to reduced cell proliferation. Apoptosis was inhibited by caspase inhibitor Q-VD-OPh or Bcl-2/xL overexpression; however, blocking apoptosis did not restore cell division. Loss of CAML did not activate the unfolded protein response, but did induce c-Jun and JNK phosphorylation. Structure-function studies indicated that lymphoma cell viability did not depend on interaction of CAML with TRC40, another component of TA protein insertion machinery. Conversely, expression of a minimal region of CAML that interacts with the TA insertion co-factor WRB was sufficient to rescue growth of CAML-deleted cells. In summary, our study demonstrates an essential pro-survival role for CAML in c-Myc-driven lymphomas. Future investigations will focus on the underlying mechanism of apoptosis in this system to advance our understanding of the biological functions of CAML, and consider CAML-dependent survival as a potential novel target for anticancer therapy.

Citation Format: Jennifer C. Shing, Lonn D. Lindquist, Richard J. Bram. c-Myc-driven lymphomas require calcium-modulating cyclophilin ligand (CAML) for survival and proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4412.