Citations

MRS provides a valuable tool for the non‐invasive detection of brain γ‐aminobutyric acid (GABA) in vivo. GABAergic dysfunction has been observed in the aging cerebellum. The study of cerebellar GABA changes is of considerable interest in understanding certain age‐related motor disorders. However, little is known about the reproducibility of GABA MRS in an aged population. Therefore, this study aimed to explore the feasibility and reproducibility of GABA MRS in the aged cerebellum at 3.0 T and to examine the effect of differing tissue composition on GABA measurements. MRI and 1H MRS examinations were performed on 10 healthy elderly volunteers (mean age, 75.2 ± 6.5 years) using a 3.0‐T Siemens Tim Trio scanner. Among them, five subjects were scanned twice to assess the short‐term reproducibility. The MEGA‐PRESS (Mescher–Garwood point‐resolved spectroscopy) J‐editing sequence was used for GABA detection in two volumes of interest (VOIs) in the left and right cerebellar dentate. MRS data processing and quantification were performed with LCModel 6.3‐0L using two separate basis sets, generated from density matrix simulations using published values for chemical shifts and J couplings. Raw metabolite levels from LCModel outputs were corrected for cerebrospinal fluid contamination and relaxation. GABA‐edited spectra yielded robust and stable GABA measurements with averaged intra‐individual coefficients of variation for corrected GABA+ between 4.0 ± 2.8% and 13.4 ± 6.3%, and inter‐individual coefficients of variation between 12.6% and 24.2%. In addition, there was a significant correlation between GABA+ obtained with the two LCModel basis sets. Overall, our results demonstrated the feasibility and reproducibility of cerebellar GABA‐edited MRS at 3.0 T in an elderly population. This information might be helpful for studies using this technique to study GABA changes in normal or diseased aging brain, e.g. for power calculations and the interpretation of longitudinal observations. Copyright © 2015 John Wiley & Sons, Ltd.

Case: We report two cases of modular head-neck junction failure involving the Stryker Accolade TMZF stem leading to sudden dissociation of the femoral head from the stem. Both patients presented with mechanical symptoms in the hip followed by pain and hip dysfunction. Disassembly of the head and deformation of the male taper were seen on preoperative radiographs. Intraoperatively, both patients had substantial metallosis with a markedly damaged taper requiring stem revision. Conclusion: We recommend regular clinical and radiographic surveillance of patients with the Stryker Accolade TMZF stem, especially those patients with pain and/or mechanical symptoms.

Abstract The t(8;21) chromosomal translocation produces AML1-ETO, an oncogenic fusion protein that compromises the function of AML1, a transcription factor critical for the myeloid cell differentiation. The differentiation arrest and self-renewal phenotypes of cells expressing AML1-ETO have been chiefly studied at the single gene level. In order to understand how the genome-wide occupancy of AML1-ETO and associated co-regulators contributes to the leukemic properties of Kasumi-1 cells, a model system for human t(8;21) leukemia, chromatin immunoprecipitation-sequencing (ChIP-seq) experiments were employed. Genome-wide DNA binding profiles for AML1 and AML1-ETO in Kasumi-1 cells showed that both proteins were enriched at introns and distal upstream regulatory regions, the latter implying that distal elements are likely important for target gene regulation. Importantly, A substantial fraction of AML1/AML1-ETO-p300 co-localization occurs near transcriptional start sites (TSSs) in promoter regions associated with transcriptionally active loci, as indicated by an observed enrichment of the H3K4me3 histone modification. A significant discovery of the current study is the co-localization of AML1/AML1-ETO with N-CoR on regions that are primarily distal to TSSs. Analyses of both gene expression data and gene ontology suggest that the AML1/AML1-ETO co-localization with N-CoR, more so than with p300, is associated with the leukemic phenotype of Kasumi-1 cells. De novo motif discovery reveals that PU.1 motifs are over-represented in regions co-occupied by AML1-ETO and N-CoR compared to other AML1-ETO bound regions. Our findings establish a novel and predominant AML leukemia signature characterized by occupancy of the AML1-ETO/N-CoR complex at genomic regions enriched in motifs for myeloid differentiation factors. We propose that this property is critical for maintenance of the leukemic phenotype. Citation Format: Daniel J. Trombly, Troy W. Whitfield, Srivatsan Padmanabhan, Jonathan A. Gordon, Jane B. Lian, Andre J. van Wijnen, Sayyed K. Zaidi, Janet L. Stein, Gary S. Stein. Co-occupancy of AML1-ETO and N-CoR defines a dominant phenotypic signature in leukemic cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B28.

Abstract The known genetic variance for epithelial ovarian cancer (EOC) accounts for less than half the familial risks of the disease. BRCA1 and BRCA2 are the major high-penetrance EOC susceptibility genes. More recently genes that interact with BRCA1 and BRCA2 in the same biological pathways, but conferring more moderate penetrance (e.g. RAD51C, RAD51D) have also been identified. Mutations in these genes result in defects in homologous recombination and double strand break repair (HR/DSB). We hypothesized that other genes operating in these and other DNA repair pathways may also confer EOC susceptibility. Whole exome sequencing data, available from more than 400 high-grade serous ovarian cancers, were used to identify 24 candidate genes harboring germline truncating mutations in at least 2 cases, followed by replication analysis using targeted resequencing in ~3,000 cases and 3,000 controls. Subjects were also sequenced for the known susceptibility genes listed above. The data confirmed the previously reported contributions of BRCA1, BRCA2, RAD51C and RAD51D to EOC. In addition, we identified BRIP1, and FANCM as candidate EOC susceptibility genes. Surprisingly, for several genes we found truncating mutations were equally prevalent in cases as they were in controls. These mutations may still represent rare low-penetrance susceptibility alleles for EOC, which we are too limited in sample size and power to confirm; or more likely, it indicates that cells can tolerate heterozygous loss-of-function mutations in apparently critical DNA repair genes without any impact on neoplastic development. These findings may have implications for risk prediction and prevention strategies for EOC and possibly for the identification of novel therapeutic targets. Citation Format: Susan J. Ramus, Honglin Song, Ed Dicks, Maria Intermaggio, Jane Hayward, Jonathan P. Tyrer, Jacek Gronwald, Susanne Krüger Kjær, Alice Wittemore, Beth Karlan, Kunle Odunsi, Usha Menon, David D. Bowtell, Ellen L. Goode, Paul D.P. Pharoah, Simon A. Gayther. Whole exome and targeted resequencing, of population based ovarian cancer cases and controls, identifies susceptibility genes for ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-CTRL-1213.

Abstract Background: The transcription factor Krüppel-like factor 5 (KLF5) is essential for normal homeostasis, stemness, and tumorigenesis. Typically, KLF5 functions as a transcriptional activator, including in esophageal epithelia where KLF5 promotes keratinocyte proliferation and migration by activating EGFR and ILK, respectively, and prevents transformation by activating Notch1. Yet, the function of KLF5 is context dependent, and mutation of the tumor suppressor p53 switches KLF5 from a transcriptional activator to a repressor of p21Waf1/Cip1 and from pro-proliferative to anti-proliferative. Interestingly, KLF5 also inhibits p53 itself, though the mechanism of this inhibition is unknown. We hypothesized that the functional switch of KLF5 from transcriptional activator to repressor involves the recruitment of a co-repressor. Methods: Primary human esophageal keratinocytes immortalized with hTERT were stably infected with inducible lentivirus containing shRNA against KLF5 or Sin3A. p53 mRNA and protein levels were assessed by real-time quantitative PCR and Western blotting, respectively, and p53 protein stability was evaluated by pulse-chase experiments. We employed ChIP across a 4 kb region on the p53 gene, from 2 kb upstream to 2 kb downstream of the transcription start site, to identify the region of KLF5 binding on p53. Promoter activity was assessed with a series of luciferase reporters containing varying lengths of p53 and its upstream regulatory region. Protein-protein interaction was examined by co-IP. Results: KLF5 knockdown significantly increased p53 both at the mRNA and protein levels. In unstressed cells p53 protein undergoes rapid degradation such that p53 levels are typically low. Yet, with KLF5 knockdown, despite increased p53 protein levels, we saw no changes in p53 protein stability. By ChIP, we identified a region of KLF5 binding to p53 between -600 to -356 bp upstream of the transcription start site. Several other KLF family members can interact with the co-repressor Sin3A, and we found that Sin3A also complexed with KLF5 in human primary esophageal keratinocytes. Knockdown of Sin3A abolished the inhibitory effect of KLF5 on p53, resulting in elevated p53 expression, growth inhibition, and increased apoptosis. Conclusion: KLF5 transcriptionally represses p53 via the co-repressor Sin3A. Thus, KLF5 promotes cell proliferation in normal keratinocytes by both activating pro-growth signals and inhibiting the brake machinery of the cell cycle. Citation Format: Yizeng Yang, Taicheng Zhou, Zhen Zong, Gwen Lomberk, Raul Urrutia, Jonathan Katz. Regulation of p53 function by KLF5. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2087. doi:10.1158/1538-7445.AM2015-2087

Abstract Background/purpose: Despite recent advances in the development of androgen deprivation therapies (ADT), prostate cancer (PCa) remains the second leading cause of cancer death in American men. Thus, there is a critical need to improve treatment outcomes for men with aggressive PCa. The purpose of this study was to identify and functionally characterize kinases that can serve as both prognostic biomarkers and therapeutic targets in PCa. Methods: To identify kinases associated with metastatic progression of PCa, we utilized high-density oligonucleotide arrays to interrogate the expression of all kinases in 545 prostatectomy samples, obtained from high-risk patients with long-term clinical follow-up (>10 years). We ranked all kinases by their fold change in expression between PCas that subsequently metastasized versus those that did not. We then characterized the top ranked kinase in preclinical PCa models with in vitro mechanistic experiments and in vivo therapeutic studies. Results: We nominated DNA-dependent protein kinase (DNAPK) as the top kinase associated with metastatic PCa progression. Interrogation of pathways associated with DNAPK knockdown in PC3, DU145, VCaP and C4-2B, identified canonical Wnt signaling as the top DNAPK-activated pathway. In PCa cells (PC3, LNCaP C.S. {LNCaP grown under charcoal-stripped serum condition}, C4-2B and LNCaP-AR) DNAPK inhibition significantly abrogates Wnt signaling and abolishes cell proliferation, migration and invasion. Interestingly, we found that DNAPK directly modulates the Wnt signaling independent of the androgen receptor (AR), highlighting the therapeutic potential of DNAPK inhibition in cancers resistant to ADT. Indeed, in VCaP xenograft models, pharmacologic inhibition of DNAPK with NU7441 demonstrated 3.4 fold (p<0.01) reduction of tumor growth at non-toxic doses. Using an independent prostate cancer tissue cohort, we validate DNAPK expression as a biomarker for metastatic disease progression (p = 6.4 e−06, hazard ratio = 2). Conclusion: In summary, we nominate DNAPK as a potential biomarker of disease progression and as a novel therapeutic target in aggressive prostate cancer. Our data suggests that DNAPK mediates PCa progression by upregulating Wnt signaling, and demonstrates that DNAPK inhibition results in significant tumor responses in PCa xenografts. Citation Format: Vishal Kothari, Jonathan F. Goodwin, Shuang Zhao, Elai Davicioni, Jeffrey R. Karnes, Robert B. Den, Rohit Mehra, Karen E. Knudsen, Felix Y. Feng. Investigating DNAPK as a biomarker and a novel therapeutic target in aggressive prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2853. doi:10.1158/1538-7445.AM2015-2853

Abstract Genome-wide association studies (GWAS) have identified several susceptibility regions that confer risk of two or more cancers. A region at 19p13 is associated with both breast and ovarian cancer risk in the general population as well as in BRCA1/BRCA2 mutation carriers. Using the iCOGs custom genotyping array, we analyzed 438 SNPs in this region in 7,435 ER-negative breast cancer cases and 42,599 controls from BCAC, and 15,347 ovarian cancer cases and 30,845 controls from OCAC and 15,252 BRCA1 mutation carriers. We identified two significant peaks of association. Peak 1, consisting of 13 highly correlated SNPs (r2>0.95), spans a 19.4kb region harboring the BABAM1, ANKLE1 and ABHD8 genes. Peak 1 SNPs are associated with serous ovarian cancer (top hit rs4808075, P = 9.2×10-20), ER-negative breast cancer (rs61494113, P = 1.1×10-13) and breast cancer in BRCA1 mutation carriers (rs61494113, P = 7.7×10-16). Peak 2 spans over 100kb and contains 7 SNPs associated with breast cancer in BRCA1 mutation carriers only (rs4464206, P = 8.9×10-8). Multiple assays were used to establish the putative functional roles of risk SNPs and candidate susceptibility genes in this region. Expression quantitative trait locus (eQTL) analyses identified significant genotype-gene expression correlations between genotype at the Peak 1 SNP rs480816 and ABHD8 expression in ovarian tumors (P = 3.0×10-5) and normal breast tissues (P = 2.8×10-3). The Peak 2 SNP, rs7246243, was significantly associated with BABAM1 expression (P = 5×10-3) in normal breast tissue only. Overexpression of BABAM1 in normal ovarian and breast cells did not modulate neoplastic phenotypes, whereas overexpression of ABHD8 induced a significant reduction in migration in both cell types in vitro. Differentially expressed genes in ABHD8 models, identified by RNAseq, were associated with cellular movement (P<1.82×10-44). Chromosome conformation capture (3C) identified an interaction between the ABHD8 promoter and a region containing 4 risk SNPs (rs4808075, rs10419397, rs56069439 and rs4808076) in breast and ovarian normal and cancer cells but only rs56069439 coincides with epigenetic enhancer marks in breast and ovarian cells. CRISPR/Cas9 genome editing was used to delete a 57bp region containing rs56069439 and expression of BABAM1, ANKLE1 and ABHD8 were measured. Taken together, these data suggest a common functional mechanism underlying risk of breast and ovarian cancer at this locus, mediated by rs56069439 variants differentially regulating expression of ABHD8, a gene involved in cell motility. Citation Format: Kate Lawrenson, Siddhartha Kar, Karoline Kuckenbaeker, Stacey Edwards, Qiyuan Li, Jonathan Tyrer, Jonathan Beesley, Kyriaki Michailidou, Susan Ramus, Alison Dunning, Fergus Couch, Alvaro Monteiro, Jacques Simard, Matthew Freedman, Douglas Easton, Georgia Chenevix-Trench, Paul Pharoah, Antonis Antoniou, Simon Gayther, OCAC, BCAC and CIMBA. Common functional mechanisms underlying pleiotropy at the 19p13.1 breast and ovarian cancer cusceptibility locus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2783. doi:10.1158/1538-7445.AM2015-2783