bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3550: Activation of a novel form of regulated necrosis to eliminate extracellular matrix detached epithelial cells
Authors: Hawk Mark A. , Buchheit Cassandra L. , Shen Luqun , Fagan Patrick , Schafer Zachary T.
Published: July 2016

The overwhelming majority of breast cancer deaths are caused by the metastasis of cancer cells from the primary tumor to distant sites in the body. For cancer cells to successfully metastasize, they must: detach from the primary tumor, move out of the original tissue into the circulatory or the lymphatic system, travel to a new site followed by arresting their movement, and then extravasate to begin colonizing a secondary site. When normal epithelial cells detach from the extracellular matrix (ECM), they induce caspase-dependent cell death which is known as anoikis. Anoikis can therefore serve as a critical barrier to metastasis as cancer cells are exposed to limited and variable matrix conditions during each step of the metastatic cascade. However, our previous studies suggest that anoikis evasion is not sufficient to protect ECM-detached cells from cell death. Using MCF10A mammary epithelial cells, we have previously shown that ECM-detachment induced metabolic changes can compromise the survival of detached cells, although the precise mechanism controlling cell death remains unclear. Here, we present data suggesting that ECM-detached cells can also be eliminated by regulated necrosis (RN), a genetically programmed, caspase-independent form of necrosis that is morphologically indistinguishable from classical necrosis. In general, the molecular mechanisms involved in RN are poorly understood. With this in mind, the current most appreciated subtype of RN, termed necroptosis, is dependent upon TNFα, RIP1K, RIP3K, MLKL, and PGAM5 to execute cell death. Our data in ECM-detached cells suggest that cells are being eliminated by a mechanism that is dependent on the kinase activity of RIP1K. Further studies have shown an increase in the expression of CYLD, a deubiquitinating enzyme that specifically removes ubiquitin chains from RIP1K, in turn stabilizing both RIP1K expression and activation. Upon analyzing other dependent executioners of necroptosis, we have strikingly found that TNFα, RIP3K, and MLKL are all dispensable for RN to occur in ECM-detachment. Furthermore, using a variety of molecular tools, such as 3D cell culture, we have found that PGAM5 is necessary for the execution of ECM-detachment induced RN. These findings uncover a novel and distinct RN pathway and highlight the need to more thoroughly understand RN as well as the mechanisms employed by ECM-detached cells to antagonize RN and promote their survival in detachment.

Citation Format: Mark A. Hawk, Cassandra L. Buchheit, Luqun Shen, Patrick Fagan, Zachary T. Schafer. Activation of a novel form of regulated necrosis to eliminate extracellular matrix detached epithelial 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 3550.
Abstract 3562: BCR/ABL increases EZH2 levels which regulates XIAP expression via miRNA-219 in chronic myeloid leukemia cells
Authors: Ikezoe Takayuki , Nishioka Chie , Yang Jing , Yokoyama Akihito
Published: July 2016

In this study, we showed that the levels of enhancer of zeste homolog 2 (EZH2) in bone marrow mononuclear cells (BMMNCs) isolated from individuals with chronic myeloid leukemia (CML) (n = 12) were greater than those in BMMNCs isolated from healthy volunteers (n = 6). Lentiviral transduction of the BCR/ABL gene in Ba/F3 cells increased EZH2 levels in parallel with phosphorylation of STAT5. Notably, chromatin immunoprecipitation assays showed that STAT5A bound to a promoter region of the EZH2 gene, resulting in an increase in the transcriptional activity of EZH2 in leukemia cells. Importantly, downregulation of EZH2 by short hairpin RNAs (shRNAs) inhibited the expression of the antiapoptotic protein XIAP and increased the miR-219 levels associated with a decrease in hypermethylation of miR-219-1 CpG islands. Moreover, overexpression of miR-219 decreased the levels of XIAP in CML cells. Since the 3′-untranslated region (3′-UTR) of XIAP contains miR219-5p-complementary binding site, miR-219 might modulate the expression of XIAP through binding of miR-219 on the 3′-UTR of XIAP.

Taken together, BCR/ABL positively regulates the expression of EZH2 via STAT5 signaling. EZH2 modulates epigenetic changes at DNA methylated regions encoding miR-219 and downregulates the level of miR-219, resulting in upregulation of XIAP.

Citation Format: Takayuki Ikezoe, Chie Nishioka, Jing Yang, Akihito Yokoyama. BCR/ABL increases EZH2 levels which regulates XIAP expression via miRNA-219 in chronic myeloid leukemia 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 3562.
Abstract 3580: Topoisomerase II mediated DNA damage generates unique classes of genome rearrangements
Authors: Gilbertson Matthew , Patel Radhika , Nitiss Karin C. , Nitiss John L.
Published: July 2016

Topoisomerase 2 (Top2) is the target of active anti-cancer agents such as etoposide and doxorubicin. These drugs interfere with the Top2 catalytic cycle and lead to trapping of the enzyme as a covalent protein: DNA complex. This trapped covalent complex is a unique DNA lesion that includes DNA strand breaks and covalent protein adducts at the site of the breaks. While Top2 mediated DNA damage is the major mechanism for tumor cell killing, it is also responsible for drug-induced translocations that can lead to secondary malignancies. We have developed several novel systems for assessing Top2-mediated genome instability using yeast as a model system. We developed repair proficient yeast strains that accumulate high levels of etoposide and other Top2 targeting drugs. We selected for loss-of-function mutations in the yeast CAN1 gene that arise following etoposide treatment, and examined the induced mutations by DNA sequencing. We identified a unique spectrum of mutations generated by etoposide in yeast. One novel class of mutational events induced by etoposide is relatively large deletions (>300 bp) and tandem duplications. Most of these etoposide-induced events are flanked by 4-8 nucleotide direct repeats. To further characterize the mechanism of Top2 induced genome instability, we took advantage of a novel Top2 allele isolated in our laboratory. This Top2 allele, termed top2AR, exhibits elevated levels of drug independent DNA cleavage in vitro, and inviability when combined with yeast mutants defective in DNA double strand break repair. We found a similar spectrum of large deletions and tandem duplications flanked by 4-8 nucleotide direct repeats. Interestingly, we also found that the recovery of large deletions and insertions was enhanced in yeast cells lacking Tdp1, a gene that participates in the repair of protein:DNA covalent complexes. Our results illustrate a novel type of genome rearrangement that is mediated by covalent Top2 DNA damage. It is noteworthy that the deletions and insertions flanked by short direct repeats have not been previously observed with other classes of DNA damaging agents. We suggest that the unique properties of these lesions may also contribute to oncogenic translocations induced by etoposide and other Top2 targeting drugs. We are currently using next-generation sequencing of etoposide treated yeast cells and cells carrying the top2AR allele to obtain a more complete picture of the types of alterations induced by Top2 mediated DNA damage.

Citation Format: Matthew Gilbertson, Radhika Patel, Karin C. Nitiss, John L. Nitiss. Topoisomerase II mediated DNA damage generates unique classes of genome rearrangements. [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 3580.
Abstract 3587: Multiplexed nuclear area and micronucleus screening identifies SKP1 as a human chromosome instability gene
Authors: Thompson Laura , Baergen Allison , Lichtensztejn Zelda , McManus Kirk
Published: July 2016

Chromosome instability (CIN) is defined as an increase in the rate at which whole chromosomes or large chromosomal fragments are gained or lost. It is hallmark of cancer that occurs frequently in both solid and liquid tumors. In addition, CIN is associated with highly aggressive tumors, the acquisition of multi-drug resistance, tumor recurrence and poor patient prognosis. Despite this, the majority of human CIN genes have yet to be elucidated, highlighting the need for studies aimed at identifying the defective genes that underlie CIN. In this study we utilized two complementary, image-based approaches capable of detecting CIN-associated phenotypes following RNAi-based silencing of candidate CIN genes. The first assay involves quantifying nuclear areas following silencing, where changes in mean nuclear area relative to controls act as a surrogate marker of CIN. The second approach monitors micronucleus (MN) formation where increases in the number of micronuclei are indicative of DNA damage or the mitotic defects that underlie CIN. These assays were employed in a high-content screen of 164 human candidate CIN genes in two unrelated cell lines, HT1080 and hTERT. In HT1080, the nuclear area and MN enumeration assays identified 88 and 96 putative CIN genes, respectively. In hTERT, the nuclear area and MN assays identified 112 and 19 putative CIN genes, respectively. Promising putative CIN genes such as SKP1 were identified and prioritized for subsequent validation based on the number of assays that identified the gene, and the strength of the CIN phenotype. Preliminary data collected through Western blotting, mitotic chromosome spreads and flow cytometry, provides evidence to support the validation of SKP1 as a bona fide human CIN gene. Identification and characterization of human CIN genes will provide critical insights into CIN and tumorigenesis, as well as identify potential targets that could be exploited in novel, precision medicine approaches for superior cancer treatment.

Citation Format: Laura Thompson, Allison Baergen, Zelda Lichtensztejn, Kirk McManus. Multiplexed nuclear area and micronucleus screening identifies SKP1 as a human chromosome instability gene. [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 3587.
Abstract 3600: Next generation high capacity DNA damage detection assay for chemotherapy and genotoxic compound screening
Authors: Sykora Peter , Woodgate Sandra , George Jay , Sobol Robert W.
Published: July 2016

The mechanism of action of many classes of chemotherapeutic agents involves either the repression of DNA repair or an increase in DNA damage. However the measurement of DNA damage levels within a cell has been notoriously difficult and current methods to asses DNA damage potential of new chemotherapeutics have major technical flaws. The single cell gel electrophoresis (SCGE) assay is a long-standing method for measuring levels of DNA damage within a cell. The principle of SCGE is that DNA damage can cause DNA strand breaks in cells. These breaks cause the relaxation of the compact highly supercoiled DNA. The application of an electric field while the cells are embedded in agarose allows damaged DNA to migrate faster than intact DNA. The more breaks in the DNA, the further it will travel in the agarose resulting in the formation of a “comet” tail, the size and length of which directly correlates to the amount of DNA damage. The SCGE method benefits from both technical simplicity and high sensitivity. The major drawback is the assay is extremely laborious, lacks appropriate controls and has poor reproducibility. We have recently overcome these drawbacks by developing a 96-well plate format of the SCGE, aptly named “CometChip”. The CometChip uses micro-pillar technology to create an agarose 96-well chip where each well has approximately 300 micro-wells used to capture individual cells. Using this technology we can incorporate multiple treatments, controls and time points on a single CometChip which can then be rapidly analyzed using a fluorescence based imaging instrument. The utility of the new technology was tested using 75 different chemical compounds considered either genotoxic, non-genotoxic or unknown. The compounds were tested on two lymphocyte cell lines with different p53 status to compare the accumulation and repair of DNA damage. We report that the CometChip gives highly reproducible and accurate results without loss of sensitivity. In the high throughput screening approach using multiple CometChip apparatus, we estimate the throughput of the assay to be approximately 10,000% greater than doing traditional slide based comet analysis. The massive increase in processivity brings new opportunity for large-scale compound screening. The increased sensitivity coupled with large sample sizes will allow researchers the option to measure minor changes in DNA damage with unparalleled accuracy.

Citation Format: Peter Sykora, Sandra Woodgate, Jay George, Robert W. Sobol. Next generation high capacity DNA damage detection assay for chemotherapy and genotoxic compound screening. [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 3600.
Abstract 3635: Performance of the Agilent D5000 and high sensitivity D5000 ScreenTape assays for the Agilent 4200 TapeStation system
Authors: Nitsche Rainer , Prieto-Lafuente Lidia , MacDonald Claire , Pechtl Isabell
Published: July 2016

The Agilent 4200 TapeStation system provides automated, fast, and reliable DNA and RNA electrophoresis for up to 96 samples using prepackaged reagents and minimal manual handling. The Agilent D5000 ScreenTape and High Sensitivity D5000 ScreenTape assays have been developed for the separation and analysis of DNA fragments from 100 bp to 5,000 bp, a size range that complements and resides between the Agilent D1000 ScreenTape and the Genomic DNA ScreenTape assays. The 4200 TapeStation system and the DNA ScreenTape assays can be used at several steps of the Next Generation Sequencing (NGS) workflow. With the emergence of methodologies, such as the use of Transposomes, NGS library sizes are tending to increase beyond 1,000 bp, even for the short-read NGS technologies. This Poster focuses on the performance of both D5000 ScreenTape assays with respect to the accuracy and precision of quantification and sizing, as well as the sensitivity of these assays. Data analysis for quantification and molarity determination was compared against the corresponding assay for the Agilent 2100 Bioanalyzer system. Additionally, performance of both the D5000 and High Sensitivity D5000 assays on the 4200 TapeStation was compared to the 2200 TapeStation system.

Citation Format: Rainer Nitsche, Lidia Prieto-Lafuente, Claire MacDonald, Isabell Pechtl. Performance of the Agilent D5000 and high sensitivity D5000 ScreenTape assays for the Agilent 4200 TapeStation system. [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 3635.
Abstract 3657: ZBTB7A is a tumor suppressor and novel therapeutic target in triple-negative breast cancer
Authors: Molloy Mary Ellen , Lewinska Monika , Liu Xue-Song , Wang Angela A. , Yuan Zhi-Min
Published: July 2016

Due to lack of targeted therapies triple-negative breast cancers (TNBC) are the deadliest form of breast cancer, raising a pressing need to develop new therapeutic strategies. Recent advances in tumor metabolism have created exciting therapeutic opportunities by unveiling many previously unrecognized therapeutic targets. Like other tumor types, TNBCs also feature elevated glucose and glutamine metabolism (Shen, L., et al. 2015; McCleland, M., et al. 2012). A better understanding of the underlying mechanisms may uncover novel therapeutic targets for TNBC. Our lab has recently discovered that ZBTB7A suppresses glycolysis by transcriptionally repressing key genes within the glycolytic pathway in various cancer types (Liu, X.S., et al. 2014). Currently, ZBTB7A is proposed as an oncogene in breast cancer (Zu, X., et al. 2011), however our data suggest that ZBTB7A acts as a tumor suppressor specifically in TNBC. The purpose of this study was to determine the potential therapeutic value of targeting ZBTB7A in TNBC. Using publicly available databases, we find that low ZBTB7A expression is associated with poor prognosis in patients with hormone receptor-negative breast cancers. Our studies corroborate with patient derived data by demonstrating that reduced expression of ZBTB7A in TNBC cell lines leads to increased proliferation and resistance to radiation and chemotherapy. Differential gene expression analysis across human breast cancer samples in TCGA database revealed an inverse correlation between ZBTB7A and glycolytic genes. Overexpression of ZBTB7A in TNBC cells leads to decreased MCT1, LDHB and GLUT3 expression at the transcript level, consistent with our published data. Modulation of ZBTB7A expression also leads to changes in lactate production and glucose consumption in TNBC cells. Results from this study support ZBTB7A as a novel tumor suppressor and perspective therapeutic target in TNBC.

Citation Format: Mary Ellen Molloy, Monika Lewinska, Xue-Song Liu, Angela A. Wang, Zhi-Min Yuan. ZBTB7A is a tumor suppressor and novel therapeutic target in triple-negative 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 3657.
Abstract 3664: RARRES2 functions as a direct tumor suppressor by promoting beta-catenin phosphorylation and degradation independent of CMKLR1 and CMKLR1-mediated antitumor immune response in adrenocortical carcinoma
Authors: Liu-Chittenden Yi , Gaskins Kelli , Wang Sophie , Kebebew Electron
Published: July 2016

Tumor suppressors and the immune system are critical players in tumor suppression. However, little is known about whether a tumor suppressor can function through both immune-dependent and immune-independent mechanisms. RARRES2 is a small secreted protein that is down-regulated in multiple human cancer types such as adrenocortical carcinoma (ACC), hepatocellular carcinoma (HCC) and melanoma. It has been shown that RARRES2 can play an indirect tumor-suppressive role by acting as a chemoattractant to recruit anti-cancer immune cells expressing its receptor CMKLR1 to sites of tumor. In this study, we show that epigenetic CpG hypermethylation of RARRES2 in vivo and in vitro in ACC causes RARRES2 down-regulation. We also found that RARRES2 can function as a direct tumor suppressor and that its tumor suppressive effect does not depend on the recruitment of CMKLR1-expressing immune cells. We show that although exogenous treatment of cultured cells with recombinant human RARRES2 protein did not show any tumor-suppressive effect, transient overexpression of RARRES2 significantly inhibited cellular invasion. Stable overexpression of RARRES2 in ACC cell line NCI-H295R led to significantly reduced cell proliferation, cell invasion, and tumorigenecity in a RARRES2 dose-dependent manner. Using xenografts of stable NCI-H295R cell lines in two immunodeficient mouse models (athymic nude mice and NSG mice), we found that in the absence of functional immune cells, stable RARRES2 overexpression can still significantly suppress tumor growth in vivo. RARRES2 overexpression promoted beta-catenin phosphorylation and degradation, reduced the phosphorylation of p38 MAP kinase and inhibited the secretion of matrix metalloprotease protein 10 (MMP10) from cancer cells in a RARRES2 dose-dependent manner. Taken into account that CMKLR1 expression is barely detectable in all the cell lines tested, our results present the first evidence that RARRES2 can function as a direct tumor suppressor independent of CMKLR1 and CMKLR1-mediated anti-tumor immune response.

Citation Format: Yi Liu-Chittenden, Kelli Gaskins, Sophie Wang, Electron Kebebew. RARRES2 functions as a direct tumor suppressor by promoting beta-catenin phosphorylation and degradation independent of CMKLR1 and CMKLR1-mediated antitumor immune response in adrenocortical carcinoma. [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 3664.
Abstract 3671: Polarity and tumor development: Regulation by adenomatous polyposis coli
Authors: Lesko Alyssa C. , Chandra Madeline , Prosperi Jenifer R.
Published: July 2016

The process of epithelial polarity is tightly regulated by multiple complexes including aPKC, PAR, and Crumbs proteins. Our laboratory has recently investigated loss of Adenomatous Polyposis Coli (APC) in the MDCK 3D culture system. We have observed a lack of hollow lumens and mislocalization of podocalyxin (gp135) to the basal surface, both of which are independent of the canonical Wnt/β-catenin signaling pathway. We further showed that the c-terminus of APC, which contains binding domains for polarity proteins (Dlg and Scrib) and cytoskeletal proteins (microtubules and actin), is responsible for the observed phenotypes. In the current study, we have investigated the molecular mechanisms required for the inversion of polarity and the potential impact on tumorigenesis. We have made the novel observation that different mechanisms are responsible for the increased size versus altered polarity observed in cysts derived from APC-KD cells. Alterations in size, although independent of changes in proliferation and/or apoptosis, are regulated by the c-terminal domain, focal adhesion kinase (FAK), Src, and β1-integrin. Despite this, the mis-localization of gp135 is only rescued by the c-terminal domain of APC, suggesting a unique molecular mechanism. As loss of epithelial polarity is an early marker for tumor development, we investigated tumor-associated phenotypes. APC-KD cells were found to grow larger colonies in soft agar, and have increased migration in wound-filling assays, which appeared to be regulated through similar molecular mechanisms. These data suggest that APC regulates polarity and associated disease phenotypes (including kidney development, cardiovascular disease, and cancer) through pathways independent of Wnt signaling.

Citation Format: Alyssa C. Lesko, Madeline Chandra, Jenifer R. Prosperi. Polarity and tumor development: Regulation by adenomatous polyposis coli. [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 3671.
Abstract 3673: BVES dependent regulation of YAP1 in colorectal cancer cells
Authors: Mittal Mukul K. , Bilotta Anthony , Keating Cody E. , Short Sarah P. , Williams Christopher S.
Published: July 2016

Colorectal cancer (CRC) has a US incidence rate of almost 150,000 cases and accounts for over 10% of new cancer diagnoses. Dysfunction of epithelial adherens junctions and tight junctions (TJs) have long been implicated in CRC growth and metastasis The Popeye domain containing family of proteins are TJ-associated and appear to nucleate TJ formation. BVES or POPDC1 is one member of this family and is under-expressed in all stages of human colorectal carcinoma (CRC). On the other hand, YAP1 a novel oncogene in the Hippo pathway is up-regulated in many cancers including colon cancer. In the present study, we report an inverse correlation between tumor suppressor protein BVES and oncogene YAP1 in human colorectal carcinoma tumor samples and human colon cancer cell lines. We demonstrate that ectopic expression of BVES in BVES deficient cells decreases YAP1 protein while knocking down BVES in cells expressing BVES increases YAP1 levels. Cycloheximide (CHX) experiments indicate that this is likely occurring at the level of protein stability. CDK6 is a cell cycle regulator and YAP1 transcriptional target, and its levels are increased in the setting of BVES-knockdown in CACO2 cells. Concurrent with this there is increased cell growth. Conversely, when BVES is overexpressed, CDK6 transcription is reduced. Further knocking down of YAP1 in BVES knocked down cells reverses the cell growth advantage suggesting that BVES regulation of cell growth in part is dependent upon YAP1 protein levels. Co-immunoprecipitation (Co-IP) and yeast-2-hybrid (Y2H) assay also shows a direct interaction of BVES and YAP1. In sum, these findings suggest a novel mechanism underlying BVES tumor-suppressive function through regulation of a potential growth modulator, YAP1 in colorectal cancer.

Citation Format: Mukul K. Mittal, Anthony Bilotta, Cody E. Keating, Sarah P. Short, Christopher S. Williams. BVES dependent regulation of YAP1 in colorectal 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 3673.
Abstract 3678: The tumor suppressive small GTPase DiRas3 (ARHI) inhibits proliferation and activation of NF-κB in glioblastoma
Authors: Rymaszewski Amy , Straza Michael , Frei Anne , Bergom Carmen
Published: July 2016

Glioblastoma (GB) is the most aggressive malignancy affecting the central nervous system (CNS) with a median survival of 12 to 15 months even with surgery, radiation and chemotherapy. Previous research demonstrates that increased activation of NF-κB is critical for GB growth, proliferation and the up regulation of genes involved in cytokine production, cell cycle regulation, apoptosis and cell adhesion. Understanding the molecular targets that regulate NF-κB may provide more effective therapeutic targets for GB. DiRas family small GTPases, which are homologous to pro-oncogenic Ras GTPases, are tumor suppressive rather than tumor promoting and include DiRas1, DiRas2 and DiRas3 (ARHI). DiRas1 and DiRas2 have been suggested to be tumor suppressive in CNS malignancies, but the role of DiRas3 in CNS malignancies remains unknown. Here we demonstrate that expression of DiRas3 protein in GB cell lines is absent, although DiRas3 is expressed in non-malignant glial cells. Re-expression of DiRas3 in U-87 cells reduces cell proliferation by 20%. Using a NF-κB transcriptional activity luciferase reporter assay demonstrates that DiRas3 expression reduces NF-κB transcriptional activity by 70% compared to vector control. Further experiments demonstrate that decreased NF- κB activity occurs via reduced phosphorylation of the NF-κB inhibitor IκBα. The reduced phosphorylation of IκBα could be a result of decreased AKT and ERK activity, as increased ERK and AKT activity can stimulate NF-κB pathways. Our lab has previously demonstrated that the most common binding partner for DiRas1 and DiRas2 was the small GTPase binding protein SmgGDS, and DiRas1 and DiRas2 also reduce NF- κB activation. However, DiRas3 does not interact with SmgGDS, suggesting that DiRas3 can reduce NF-κB in a SmgGDS-independent manner. Understanding the role of DiRas3 and its binding partners in mediating NF- κB activation may lead to novel therapeutics for glioblastoma.

Citation Format: Amy Rymaszewski, Michael Straza, Anne Frei, Carmen Bergom. The tumor suppressive small GTPase DiRas3 (ARHI) inhibits proliferation and activation of NF-κB in glioblastoma. [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 3678.
Abstract 3686: Elephant TP53 (ep53) retrogene protein expression enhances cellular apoptosis in response to DNA damage
Authors: Abegglen Lisa M. , Robinson Rosann , Donovan Lauren , Caulin Aleah F. , Goldfeder Mor , Schroeder Avi , Maley Carlo C. , Schiffman Joshua D.
Published: July 2016

We previously reported that compared with other mammalian species, elephants have a lower than expected rate of cancer (Abegglen JAMA 2015). While exploring cancer resistance in elephants, we discovered that elephants have 20 copies of TP53 (1 ancestral gene with introns and 19 retrogenes) compared to humans with 1 copy. TP53 amplification was associated with increased p53-mediated apoptosis induced by DNA damage in elephant cells compared to human cells. As a follow up to this study, we further explored the function of these elephant TP53 retrogenes by expressing their protein (ep53) in cells with 2 functional TP53 alleles. NIH/3T3 cells were transfected with a mammalian expression construct encoding TP53 retrogene 9 fused to an epitope tag (myc). Western blot analysis probing for myc confirmed expression of retrogene 9. To measure the functional consequence of expression, we compared apoptosis of cells transfected with empty vector to cells transfected with myc-tagged retrogene 9 after doxorubicin treatment. Apoptosis was measured with a triplex assay that assesses viability, cytotoxicity and apoptosis (Apo Tox-Glo, Promega). We observed a significant increase in apoptosis of NIH/3T3 cells expressing retrogene 9 compared to empty vector transfected control cells (10μM doxorubicin, P<0.001). These results suggest that ep53 is a functional protein contributing to cancer resistance in elephants, and that elephant retrogenes can function in cells from other species to enhance their apoptotic response to DNA damage. These findings may hold significance for human cancer prevention and treatment.

Citation Format: Lisa M. Abegglen, Rosann Robinson, Lauren Donovan, Aleah F. Caulin, Mor Goldfeder, Avi Schroeder, Carlo C. Maley, Joshua D. Schiffman. Elephant TP53 (ep53) retrogene protein expression enhances cellular apoptosis in response to DNA damage. [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 3686.
Abstract 369: Automating recovery of spiked tumor cells from blood
Authors: Sarinana Mark , Truong Ky , Cannon Keith , Guia Antonio
Published: July 2016

The isolation and study of circulating tumor cells (CTC) enables personalized medicine solutions, monitoring of therapeutics (theragnostics), and monitoring of minimal residual disease (MRD); culturing these rare cells is a frequent goal in cancer research, however the common methods for their isolation (gradient centrifugation, lysis buffer) are damaging to these cells, and many emerging technologies leave the cells bound in a consumable, bound to a biomarker or in a state that is difficult to use in cell culture. Further, the anuclear cells in blood greatly outnumber nucleated cells and present significant challenges to the study of the nucleated cells, for example, erythrocytes can absorb excitation or emission wavelengths in fluorescence studies, and their shape promotes rouleaux formation which can sequester the cells of interest; platelets also have a tendency to be activated by physical handling and cause aggregation that can also sequester and damage the cells of interest, and these all contain the greatest content of RNA and other markers that increase background noise in many assays. We demonstrate a new technology that automates the depletion of erythrocytes, thrombocytes, and plasma markers from, initially, small volumes of blood, using a micromachined membrane filter. We spiked small counts (10 to 1000 estimated from dilution) of tumor cell lines BT474 (breast cancer), DLD1 (colorectal cancer), and K562 (myelogenous leukemia) and other cell types into aliquots of blood. Using calcein-AM-loading to distinguish the spiked cells from blood cells, the recovery rates were determined in a flow cytometer, indicating an average of 80% ±4% (SE, N = 27) recovery of the rare cells after depletion of anuclear cells and plasma. The technology is amenable to use of beads for initial negative depletion of common nucleated cells by magnetic beads and has a recovery capacity of a few to as much as 3 million cells per specimen. Tumor cell lines alone that were run through this automation process then seeded for cell culture demonstrated growth rates indistinguishable from those seeded in similar counts in a regular cell passage. In further studies we demonstrate depletion of leukocytes with magnetic beads and of anuclear cells by filtration to handle larger blood volumes. The filtration system may be used for automated recovery of nucleated cells or cell clusters from blood or other fluid samples (liquid biopsies) without use of harsh chemicals or centrifugation, removing a common barrier for emerging analytical approaches.

Citation Format: Mark Sarinana, Ky Truong, Keith Cannon, Antonio Guia. Automating recovery of spiked tumor cells from blood. [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 369.
Abstract 3700: Association of the G/G-SNP309 variant in the mdm2 gene with earlier tumor onset in female renal cell carcinoma patients
Authors: Stoehr Christine G. , Stoehr Robert , Wenners Antonia , Hartmann Arndt , Bertz Simone , Spath Verena , Walter Bernhard , Junker Kerstin , Moch Holger , Hintze Raoul , Denzinger Stefan , Bond Elisabeth E. , Bond Gareth L. , Bluemke Karen , Weigelt Katrin , Lieb Verena , Nolte Elke , Fornara Paolo , Wullich Bernd , Wach Sven , Taubert Helge W.
Published: July 2016

Mdm2 (Human mouse double minute 2) is an important opponent of the tumor suppressor p53. The G/G variant of SNP309 in the MDM2 promoter can increase Mdm2 mRNA/protein expression and is associated with an increased risk and earlier onset of different cancers especially in Asian populations. But the frequency and impact of the G/G variant has not been studied in Caucasian renal cell carcinoma (RCC) patients. Therefore, we analyzed an unselected German cohort of 197 consecutive RCC patients and detected the G/G variant in 18 (9.1%) patients, the G/T variant in 116 (58.9%) patients and the T/T variant in 63 (32.0%) patients. Studying the association between age at tumor onset and SNP309 genotypes, no correlation was detected in the entire RCC cohort or among the male RCC patients. However, the female G/G patients (median age 59.5 years)

were diagnosed 13.5 years earlier than the T/T females (median age 73 years). Next, when separating all females into two groups at their median age (68 years), 7 patients and 1 patient with the G/G variant and 9 patients and 13 patients with the T/T variant were noted in these age groups (P = 0.024). To study the age dependency of tumor onset further, a second, age-selected cohort of 205 RCC patients was analyzed, which comprised especially young and old patients. Interestingly, the G/G type occurred more often at lower tumor stages and tumor grades compared with higher stages (P = 0.039 and P = 0.004, respectively). In females, the percentage of the G/G variant was only slightly higher in the younger age group, whereas in males, the percentage of the G/G variant was remarkably higher in the younger age group (19.4% vs. 8.0%). In summary, female Caucasian RCC patients with the MDM2 SNP 309 G/G variant showed a significantly earlier tumor onset than patients with the wildtype T/T genotype.

Citation Format: Christine G. Stoehr, Robert Stoehr, Antonia Wenners, Arndt Hartmann, Simone Bertz, Verena Spath, Bernhard Walter, Kerstin Junker, Holger Moch, Raoul Hintze, Stefan Denzinger, Elisabeth E. Bond, Gareth L. Bond, Karen Bluemke, Katrin Weigelt, Verena Lieb, Elke Nolte, Paolo Fornara, Bernd Wullich, Sven Wach, Helge W. Taubert. Association of the G/G-SNP309 variant in the mdm2 gene with earlier tumor onset in female renal cell carcinoma patients. [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 3700.
Abstract 3716: Potent radiation enhancement with VX-984, a selective DNA-PKcs inhibitor for the treatment of NSCLC
Authors: Boucher Diane , Hoover Russell , Wang Yuxin , Gu Yong , Newsome David , Ford Pamella , Moody Cameron , Damagnez Veronique , Arimoto Reiko , Hillier Shawn , Wood Mark , Markland William , Eustace Brenda , Cottrell Kevin , Penney Marina , Furey Brinley , Tanner Kirk , Maxwell John , Charifson Paul
Published: July 2016

Ionizing radiation (IR), which is widely used for the treatment of cancer, causes double-strand breaks (DSBs) in DNA. If left unrepaired, these DSBs are lethal to the cell. DNA-dependent protein kinase (DNA-PK) is a key enzyme in the non-homologous end joining (NHEJ) pathway that repairs DSBs caused by IR, or chemotherapeutic agents that cause DSBs such as doxorubicin. The goal of these studies was to characterize the radiation enhancing effects of VX-984, a selective and potent ATP-competitive inhibitor of the catalytic subunit of DNA-PK (DNA-PKcs), with a focus on non-small cell lung cancer (NSCLC) cells and tumor xenografts. VX-984 enhances the cytotoxicity of IR in a panel of cancer cell lines including NSCLC cell lines in vitro with dose enhancement factors (DEF) greater than 3. Notably, VX-984 combined with IR in normal human lung fibroblasts minimally enhanced the cytotoxicity compared to IR alone. Additionally, VX-984 decreased DNA-PKcs autophosphorylation on S2056 both in vitro and in vivo in NSCLC cells and attenuated the decay of the DNA damage markers γH2AX and pKAP1 in response to IR. In NSCLC PDX models VX-984, in combination with IR (2 Gy x 3), caused durable complete responses while IR alone only led to a delay in tumor growth, consistent with delayed DNA damage repair. In these models, the combination of VX-984 and IR was well tolerated. These data demonstrate that VX-984 is a potent radiation-enhancing agent and provide a strong rationale for the use of VX-984 in combination with IR for the treatment of NSCLC.

Citation Format: Diane Boucher, Russell Hoover, Yuxin Wang, Yong Gu, David Newsome, Pamella Ford, Cameron Moody, Veronique Damagnez, Reiko Arimoto, Shawn Hillier, Mark Wood, William Markland, Brenda Eustace, Kevin Cottrell, Marina Penney, Brinley Furey, Kirk Tanner, John Maxwell, Paul Charifson. Potent radiation enhancement with VX-984, a selective DNA-PKcs inhibitor for the treatment of NSCLC. [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 3716.
Abstract 3724: Combination of TRAIL and Chal-24 synergistically kills lung cancer cells through autophagy-mediated degradation of IAPs and c-FLIPL
Authors: Xu Jennings , Xu Xiuling , Shi Shaoqing , Wang Qiong , Saxton Bryanna , Xing Chengguo , Lin Yong
Published: July 2016

Combination chemotherapy is an effective strategy for increasing anticancer efficacy, reducing side effects and alleviating drug resistance. Here we report that combination of the recently identified novel chalcone derivative, chalcone-24 (Chal-24), and TNF-related apoptosis-inducing ligand (TRAIL) significantly increases cytotoxicity in lung cancer cells. Chal-24 treatment significantly enhanced TRAIL-induced activation of caspase-8 and caspase-3, and the cytotoxicity induced by combination of these agents was effectively suppressed by the pan-caspase inhibitor z-VAD-fmk. Chal-24 and TRAIL combination suppressed expression of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein large (c-FLIPL) and cellular inhibitor of apoptosis proteins (c-IAPs), and ectopic expression of c-FLIPL and c-IAPs inhibited the potentiated cytotoxicity. In addition, TRAIL and Chal-24 cooperatively activated autophagy. Suppression of autophagy effectively attenuated cytotoxicity induced by the Chal-24 and TRAIL combination, which was associated with attenuation of c-FLIPL and c-IAPs degradation. Altogether, these results suggest that Chal-24 potentiates the anticancer activity of TRAIL through autophagy-mediated degradation of c-FLIPL and c-IAPs, and that combination of Chal-24 and TRAIL could be an effective approach in improving chemotherapy efficacy.

Citation Format: Jennings Xu, Xiuling Xu, Shaoqing Shi, Qiong Wang, Bryanna Saxton, Chengguo Xing, Yong Lin. Combination of TRAIL and Chal-24 synergistically kills lung cancer cells through autophagy-mediated degradation of IAPs and c-FLIPL. [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 3724.
Abstract 3761: The MDM2 inhibitor AMG 232 causes tumor regression and potentiates the anti-tumor activity of MEK inhibition and DNA-damaging cytotoxic agents in preclinical models of acute myeloid leukemia
Authors: Canon Jude R. , Osgood Tao , Saiki Anne Y. , Oliner Jonathan D.
Published: July 2016

AMG 232 is a potent inhibitor of the MDM2-p53 interaction and is a promising clinical candidate for treating tumors, in particular those harboring wild-type p53. AML represents a compelling indication for AMG 232 given the low rate of p53 mutation, frequent MDM2 overexpression, and unmet medical need. We evaluated the effect of AMG 232 treatment on AML tumors in vitro and in vivo, elucidated the mechanism of anti-tumor efficacy, and tested the effect of combining AMG 232 with targeted agents and chemotherapeutics. Combinations were identified based on evidence of in vitro synergy from cell based screens, or based on biological rationale and clinical opportunity with standard of care agents. Combinations evaluated included MEK inhibitors, and p53-inducing, DNA-damaging cytotoxics cytarabine, doxorubicin, and decitabine. In vitro assays demonstrated that AMG 232 as a single agent was effective at inducing cell death across a panel of p53 wild-type AML cell lines. The anti-tumor efficacy involved activation of the p53 pathway, robust inhibition of the cell cycle and induction of apoptosis. AMG 232 treatment caused AML tumor regression in vivo which was related to dose- and time-dependent induction of the p53 targets p21 and PUMA. The combination of AMG 232 and MEK inhibition resulted in synergistic tumor cell killing in vitro, and enhanced in vivo anti-tumor activity which was significantly better than either single agent. Combinations of AMG 232 with chemotherapies which induce DNA damage resulted in synergistic in vitro cell killing, and superior anti-tumor efficacy in vivo with increased induction of p53 signaling in tumors. These data support a clinical strategy for evaluating AMG 232 as a monotherapy and in combination with targeted and cytotoxic agents to treat AML patients.

Citation Format: Jude R. Canon, Tao Osgood, Anne Y. Saiki, Jonathan D. Oliner. The MDM2 inhibitor AMG 232 causes tumor regression and potentiates the anti-tumor activity of MEK inhibition and DNA-damaging cytotoxic agents in preclinical models of acute myeloid leukemia. [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 3761.
Abstract 3795: Novel and selective MELK kinase inhibitors active in breast cancer cell lines
Authors: Carpinelli Patrizia , Montemartini Marisa , Amboldi Nadia , Ballinari Dario , Cribioli Sabrina , Ciomei Marina , Colombo Riccardo , Re Depaolini Stefania , Avanzi Nilla , Canevari Giulia , Ceccarelli Walter , Posteri Helena , Brasca Maria Gabriella , Donati Daniele , Felder Eduard Rudolf , Isacchi Antonella , Galvani Arturo , Montagnoli Alessia
Published: July 2016

Maternal Embryonic Leucine zipper Kinase (MELK) is a serine-threonine kinase implicated in stem cell renewal, override of cell cycle checkpoints, pre-mRNA splicing and resistance to apoptosis, while MELK gene expression levels correlate inversely with poor prognosis in breast cancer, prostate cancer and glioblastoma patients. Moreover, recent findings underlie the oncogenic role of this kinase in triple negative breast cancer (TNBC), a category of high-grade, invasive tumors which lack expression of estrogen receptor (ER) and progesterone receptor (PR) and HER2 amplification and which is resistant to current cytotoxic and targeted therapies. Furthermore, they are highly heterogeneous with respect to genomic alterations, and common therapeutic targets are lacking, although substantial evidence implicates dysregulated kinase signaling.

Here, we describe the preclinical characterization of novel, potent and selective ATP-competitive MELK kinase inhibitors identified by means of high-throughput screening of the NMS proprietary compound collection. Leading compounds possess biochemical activity against MELK in the nanomolar range with high selectivity against a panel of 60 further kinases representative of the human kinome. Amongst human tumor cell lines tested in 2-dimensional colony outgrowth assays, marked sensitivity was observed in breast cancer cell lines, with sub-micromolar anti-proliferative activity. This effect was accompanied by dose-dependent induction of apoptosis and by modulation of cellular biomarkers, consistent with a MELK-dependent mechanism of action.

Overall, these data provide further evidence that MELK is a promising biological target for the development of novel anticancer therapies.

Citation Format: Patrizia Carpinelli, Marisa Montemartini, Nadia Amboldi, Dario Ballinari, Sabrina Cribioli, Marina Ciomei, Riccardo Colombo, Stefania Re Depaolini, Nilla Avanzi, Giulia Canevari, Walter Ceccarelli, Helena Posteri, Maria Gabriella Brasca, Daniele Donati, Eduard Rudolf Felder, Antonella Isacchi, Arturo Galvani, Alessia Montagnoli. Novel and selective MELK kinase inhibitors active in breast cancer cell lines. [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 3795.