bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A30: NF-kB activating peptides identified in genetic screen cooperate with Ras transformation by suppression of p53 linking inflammation to carcinogenesis
Authors: Natarajan Venkatesh , Komarov Andrei P. , Chenchik Alex , Gudkov Andrei V.
Published: August 2014

Chronic inflammatory conditions are frequently linked to increased risk of carcinogenesis. The transcription factor NF-kappa B acts as a central component of signaling pathways governing the inflammatory response. NF-kB is also constitutively activated in several tumors. While the tumor promoting inflammatory effects are largely attributed to activation of NFκB signaling in tumor milieu, it is unclear whether activation of inflammatory pathway alone can initiate tumorigenesis. The mechanism by which constitutive activation of NF-kB contributes to carcinogenesis remains elusive. We hypothesized that constitutive activation of NFkB by proinflammatory agents renders p53 incapable of opposing oncogene induced transformation. As pharmacological activation of NF-kB in normal cells is prone to transient effects due to feedback regulation, we performed an unbiased genetic screen for proinflammatory peptides in an NFκB-GFP reporter cell line using a lentiviral trimer peptide library spanning the coding regions of 800 extracellular ligands. We identified several pro-inflammatory peptides whose expression lead to robust activation of NFκB signaling. Suppression of p53 has been shown to be the only criteria to be met to override ras oncogene induced senescence in rodent fibroblasts, which prompted us to test the NFκB activating peptides in this model. Here, we show that indeed, these NF-kB activating peptides cooperate with oncogenic RasV12 to induce transformation in both in rat fibroblast cell line REF52 and primary mouse embryonic fibroblasts (MEFs) through r p53. Further, these Ras transformed fibroblast cells formed tumors in nude mice. Thus, pro-inflammatory genetic elements that activate NF-kappa B pathway can override Ras induced senescence to facilitate cellular transformation underscoring the importance of NFκB inhibitors in preventing initiation of tumerigenesis.

Citation Format: Venkatesh Natarajan, Andrei P. Komarov, Alex Chenchik, Andrei V. Gudkov. NF-kB activating peptides identified in genetic screen cooperate with Ras transformation by suppression of p53 linking inflammation to carcinogenesis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A30.
Abstract A33: Optimizing NGS-based shRNA screening experiments and data analysis
Authors: Yu Jiyang , Gao Ying , Zhong Wenyang , Chen Lei , Apanovitch Donald , Follettie Max , Rejto Paul , Arndt Kim
Published: August 2014

Pooled small hairpin RNA (shRNA) screening emerges as a powerful tool for loss-of-function studies in mammalian cells and searching for novel or synthetic lethality-based therapeutic targets of human tumors. Next-generation sequencing (NGS) is becoming a standard technology to read out hairpin representations with high resolution and allowance of massive multiplexing. However, the extraction of robust biological signals from high-throughput NGS-based shRNA screening (shSeq) data remains challenging due to low knock-down efficacy and off-target effects of shRNAs, sequencing errors, artificial mutations, etc. In this presentation, based on our in vitro shSeq screens in human cancer cells by Illumia MiSeq, we report a discovery of an unexpected recurrent distribution pattern of identified hairpin counts across number of allowed mismatches during read deconvolution. By exhaustive analysis of within and between-group noise, we develop a procedure to optimize both experimental and analytical parameters for shSeq screens and suggest a set of default parameters that perform well consistently. We observe that over 10% of recovered hairpin reads have at least one mismatch from the reference sequence, which cannot be explained by sequencing error only. We also notice that there are more mismatched reads at later time point than at initial one. And computational analysis of such dynamic process reflects higher evolutionary mutation rate (>10^-5) in cancer cells than common sense (~10^-9). Artificial mutations that might cause a high percentage of mismatches are further explored and interesting mutation patterns are identified.

Citation Format: Jiyang Yu, Ying Gao, Wenyang Zhong, Lei Chen, Donald Apanovitch, Max Follettie, Paul Rejto, Kim Arndt. Optimizing NGS-based shRNA screening experiments and data analysis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A33.
Abstract A41: Exploiting synthetic lethal interactions in the DNA damage response area and beyond in drug discovery
Authors: Wappett Mark , Ward Richard , Fisher Paul , Bradford James , Dry Jonathan , O'Connor Mark , Lau Alan , Durant Steve
Published: August 2014

Building upon the exciting precedent exemplified by olaparib in the PARP-BRCA axis, we aim to identify and exploit further synthetic lethal (SL) interactions within and beyond the DNA damage response (DDR) area. To facilitate with this search, we have developed a method implemented as an in silico algorithm that uses gene expression and mutation data to detect putative SL interactions in cell lines and cancer tissue across The Cancer Genome Atlas (TCGA). This approach has detected relatively rare signatures including those described in the literature, for example ATM and ATR. The algorithm has also revealed potentially novel in silico SL gene pairs outside of DDR involving frequently mutated tumor suppressor genes linked to targets that AstraZeneca has previously developed selective compounds against. We are validating an attractive selection of these in vitro using appropriate cell lines, siRNA and small molecule inhibitors.

Citation Format: Mark Wappett, Richard Ward, Paul Fisher, James Bradford, Jonathan Dry, Mark O'Connor, Alan Lau, Steve Durant. Exploiting synthetic lethal interactions in the DNA damage response area and beyond in drug discovery. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A41.
Abstract B05: Targeting nonapoptotic cell death in chemoresistant patient-derived breast cancer cells
Authors: Vaden Rachel M. , Gligorich Keith M. , Shelton Dawne N. , Wang Guoying , Matsen Cindy B. , Looper Ryan E. , Sigman Matthew S. , Welm Bryan E.
Published: August 2014

The development of anti-cancer therapeutics that target non-apoptotic pathways has received increasing attention from the scientific community in recent years. The prevalence of chemoresistance and the characterization of defective apoptotic pathways in cancer has promoted interest in the expansion of current therapeutic regimens to include drugs that induce non-apoptotic cell death. From a phenotypic screen we have identified a small molecule named C6 that induces caspase-independent, non-apoptotic cell death in chemoresistant patient-derived breast cancer cells. Additionally, C6 is also selectively cytotoxic against cancer cells compared to normal mammary epithelial cells. In an effort to characterize this small molecule's mechanism of action and identify relevant biological pathways that might be used as therapeutic drug targets, we have utilized a photoaffinity pull-down strategy to identify biological binding partners of C6. Our photoaffinity pull-down studies have revealed Mitsugumin 23 (MG23), an endoplasmic reticulum-bound transmembrane protein capable of ion channel formation, as a binding partner for C6. Additionally, we have identified a metabolic component of C6-induced cell death through the use of mitochondrial respiration measurements, metabolomic analyses, and mitochondrial transmission electron microscopy (TEM) imaging. Metabolic studies have identified mitochondrial respiration defects, excess lactic acid production, and gross changes in mitochondrial morphology as a result of C6 treatment. Collectively, our findings suggest a role for ionic imbalance and subsequent metabolic disruption in this form of caspase-independent cell death. Our ongoing work is focused on further mechanistic characterization of this non-apoptotic pathway as a potential target for breast cancer drug development.

Citation Format: Rachel M. Vaden, Keith M. Gligorich, Dawne N. Shelton, Guoying Wang, Cindy B. Matsen, Ryan E. Looper, Matthew S. Sigman, Bryan E. Welm. Targeting nonapoptotic cell death in chemoresistant patient-derived breast cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B05.
Abstract B18: High-dimensional combinations of cancer drugs to induce synthetic lethality in colorectal cancer
Authors: Horn Thomas , Zhang Xiaobin , Ho Samuel , Garraway Levi , Schlegel Robert , Caponigro Giordano , Lehar Joseph
Published: August 2014

Cancers are inherently robust biological systems with many compensation mechanisms and genetic redundancies allowing them to escape treatments and become resistant. Theoretically, such complex systems can become fragile when perturbed by multiple attacks. Here, we systematically tested whether pairwise combinations of compounds targeting critical oncogenic pathways in colorectal cancer cells can combine to inhibit their growth or even kill them. Some combinations targeting growth pathways showed synergies in a broad spectrum of genetic backgrounds even when the single agents had more specific effects. Guided by the pairwise combination screening, we identified higher-dimensional combinations that not only showed broad synergy but could also drive cytostatic combination responses into synthetic lethality.

Citation Format: Thomas Horn, Xiaobin Zhang, Samuel Ho, Levi Garraway, Robert Schlegel, Giordano Caponigro, Joseph Lehar. High-dimensional combinations of cancer drugs to induce synthetic lethality in colorectal cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B18.
Abstract B25: Inhibition of autocrine signaling as a strategy to target tumor-initiating breast cancer cells
Authors: Scheel Christina , Dragoi Diana , Eaton Elinor Ng , Sophia Hsin-Jung Li , Reinhardt Ferenc , Weinberg Robert A.
Published: August 2014

The epithelial-mesenchymal transition (EMT), a pleiotropic cellular program, has been associated with the acquisition of metastatic ability, self-renewal traits and resistance to chemotherapeutic drugs in breast cancer and other carcinomas. During normal development and tumor progression, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment, thereby promoting cellular heterogeneity. The signaling context responsible for inducing an EMT and maintaining the resulting cellular states has been unclear.

We describe three signaling pathways, involving transforming growth factor (TGF)-beta and canonical/beta-catenin dependent and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal and stem cell-like state. Importantly, the induction of the EMT program and associated autocrine signaling is partly enabled by the downregulation of endogenous inhibitors of autocrine signals secreted by epithelial cells prior EMT, indicating a homeostatic mechanism. These inhibitors include secreted frizzled-related protein 1 (SFRP1), which has been shown by others to be a rate-limiting determinant of Wnt signaling, and Bone Morphogenetic Proteins, which appear to inhibit TGF-beta signaling. Conversely, disruption of autocrine signaling by adding back endogenous inhibitors of autocrine signaling as recombinant proteins inhibits migration and self-renewal of breast cancer cells in vitro and reduces tumorigenicity and spontaneous metastasis in vivo.

Together, our results indicate that ongoing autocrine signaling is required for the maintenance of mesenchymal and stem cell traits in breast cancer cells. In the longer term, we intent to develop a cocktail small-molecule inhibitors to therapeutically interrupt multiple autocrine pathways, thereby forcing tumor-initiating cells of breast and other carcinomas to exit the mesenchymal, stem cell-like state. Since this cellular state is associated with resistance to chemotherapeutic drugs, we predict that such a treatment protocol might render breast cancer cells that have passed through an EMT more sensitive to standard chemotherapy, thereby serving as a therapeutic strategy to overcome tumor heterogeneity and relapse.

Citation Format: Christina Scheel, Diana Dragoi, Elinor Ng Eaton, Sophia Hsin-Jung Li, Ferenc Reinhardt, Robert A. Weinberg. Inhibition of autocrine signaling as a strategy to target tumor-initiating breast cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B25.
Abstract B27: Probing synthetic lethal interactions with RAF inhibition in BRAF-mutant colorectal cancer
Authors: Whittaker Steven R. , Luo Flora , Hsiao Jessica , Cowley Glenn S. , Root David E. , Garraway Levi A.
Published: August 2014

Large-scale RNA interference screens have indicated that BRAF-mutant colorectal cancer (CRC) cell lines are dependent upon BRAF expression for proliferation. However, small molecule RAF inhibitors, such as vemurafenib, have so far failed to exhibit robust activity in clinical trials of patients with CRC, with a response rate of only 5%. Commensurate with this, CRC cell lines demonstrate intrinsic resistance to RAF inhibitors. Therefore, we designed an RNA interference screen to identify loss of function events that could synergize with pharmacologic RAF inhibition. We transduced a pooled lentiviral library encoding 90,000 shRNAs targeting over 16,500 genes into RAF-inhibitor-resistant RKO CRC cells. The shRNA-infected population was divided into two experimental arms, one treated with DMSO, the other treated with PLX4720. Following 16 population doublings, the abundance of each hairpin was assessed by PCR amplification of barcoded hairpin DNA, followed by massively parallel paired-end sequencing. The log-fold change in shRNA abundance between the PLX4720- and DMSO-treated controls was determined and RNAi gene enrichment (RIGER) used to rank individual shRNAs and nominate candidate genes that were required for survival of RKO cells exposed to PLX4720. We validated the 40 top-ranking genes that displayed synthetic lethality with RAF inhibition in an arrayed secondary screen. shRNAs targeting MET scored highly, consistent with recent reports implicating HGF-MET signaling in resistance to BRAF inhibition, validating our approach. Furthermore, we describe the characterization of additional genes that sensitize cells to inhibition of BRAF, suggesting novel therapeutic opportunities. By integrating these findings with other genomic and functional studies we aim to identify clinically actionable events that cause RAF-inhibitor resistance in colorectal cancers.

Citation Format: Steven R. Whittaker, Flora Luo, Jessica Hsiao, Glenn S. Cowley, David E. Root, Levi A. Garraway. Probing synthetic lethal interactions with RAF inhibition in BRAF-mutant colorectal cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B27.
Abstract B36: The role of dUTPase in ribonucleotide reductase-driven tumorigenesis and tumor progression
Authors: Chang Zee-Fen
Published: August 2014

Regulation of enzymes in nucleotide synthesis plays a critical role in DNA replication and repair. Ribonucleotide reductase (RNR) is the key enzyme that controls the de novo synthesis of all four dNTPs, and the expression of RRM2 subunit of RNR is often elevated in malignant tumor cells. Our previous study has demonstrated that RNR contributes to dUTP mis-incorporation in DNA repair when dTTP supply is deficient. In this study, we investigated the relationship between the expression level of RRM2 and dUTP pyrophosphatase(dUTPase) and tumor progression. Immunohistochemistry staining of surgical tumors from colorectal cancer (CRC) patients showed that high RRM2/low dUTPase correlates with poorer overall survival. We further analyzed the interplay of RRM2 and dUTPase in genome stress and cellular transformation. Our results revealed that constitutive elevation of RRM2 in normal fibroblasts promotes replication stress, as indicated by 53BP foci, and cellular transformation, both of which are prevented by co-expression of dUTPase. Knockdown of dUTPase in cancer cells containing high levels of RRM2 and dUTPase also increased replication stress. We proposed that the context of high RRM2/low dUTPase confers cancer cells replication stress to drive tumorigenesis and might serve as a prognostic indicator.

Citation Format: Zee-Fen Chang. The role of dUTPase in ribonucleotide reductase-driven tumorigenesis and tumor progression. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B36.
Abstract IA11: Identification of therapeutic targets for MYC-driven cancers by functional genomics
Authors: Toyoshima Masafumi , Jang In Sock , Cermelli Silvia , Bernard Brady , Grandori Carla
Published: August 2014

The development of small molecules targeting MYC oncoproteins specifically in cancer cells has been a challenge, as MYC are DNA binding proteins required for proliferation of normal tissues. To circumvent these difficulties, we identified synthetic lethal interactions with MYC over-expression by high throughput siRNA screening (Toyoshima et al, 2012).

Here, we will present validation of specific MYC-synthetic lethal genes (MYC-SL) in MYCN amplified neuroblastoma and ovarian cancer with high level of MYC using both RNAi approaches as well as small molecule inhibitors. To prioritize genes for future drug development, we carried out meta-analysis of genomic tumor data and obtained statistical significant associations between MYC alterations (i.e. gene amplification or overexpression) for selected MYC-SL genes. We also addressed the need of identifying biomarkers to stratify patients with solid tumors for activation of MYC and we will discuss the challenges and potential strategies, with particular focus on ovarian and breast cancers.

Finally, to obtain a more global view of critical pathways that MYC-SL genes we performed network analysis of our targets. This analysis revealed that major points of vulnerabilities are linked to transcription and chromatin remodeling processes, DNA repair and cell-cycle checkpoints and metabolic functions. In addition, developmental, signaling and differentiation pathways represent unforeseen dependencies of MYC overexpressing cells. Altogether, these results provide a broad menu for therapeutic interventions to target MYC-driven cancers.

*Current affiliation: Tohoku University School of Medicine, Sendai, Japan.

Citation Format: Masafumi Toyoshima, In Sock Jang, Silvia Cermelli, Brady Bernard, Carla Grandori. Identification of therapeutic targets for MYC-driven cancers by functional genomics. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr IA11.
Abstract IA14: Harnessing genetic dependencies in cancer therapy
Authors: Ashworth Alan
Published: August 2014

The design of targeted therapeutic strategies for cancer has largely been driven by the identification of tumor-specific genetic changes. However, the large number of genetic alterations present in tumor cells means that it is difficult to discriminate between genes that are critical for maintaining the disease state and those that are merely coincidental. Even when critical genes can be identified, directly targeting these is often challenging. We have exploited principles utilized for many years in model organisms to address these issues. Here I will describe particular examples of the use of synthetic lethality in cancer therapy. In addition, I will discuss high-throughput screening strategies to identify genetic dependencies in tumors.

Citation Format: Alan Ashworth. Harnessing genetic dependencies in cancer therapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr IA14.
Abstract IA16: Large-scale systematic explorations of single and combined drug responses to suggest anticancer drugs and overcome resistance
Authors: Lehar Joseph
Published: August 2014

Novartis is undertaking a large-scale effort to comprehensively describe cancer through the lens of cell cultures and tissue samples. In collaboration with academic and industrial partners, we have generated mutation status, gene copy number, and gene expression data for a library of 1,000 cancer cell lines, representing most cancer lineages and common genetic backgrounds. Most of these cell lines have been tested for chemosensitivity against ~1,200 cancer-relevant compounds, and we are systematically exploring drug combinations for synergy against ~100 prioritized CCLE lines. We expect this large-scale campaign to enable efficient patient selection for clinical trials on existing cancer drugs, reveal many therapeutically promising drug synergies or anti-resistance combinations, and provide unprecedented detail on functional interactions between cancer signaling pathways. I will discuss early highlights of this work and describe our plans to make use of this resource.

Citation Format: Joseph Lehar. Large-scale systematic explorations of single and combined drug responses to suggest anticancer drugs and overcome resistance. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr IA16.
Abstract PR02: Targeting altered SWI/SNF function through synthetic lethal RNAi screening
Authors: Conery Andrew R. , Neiss Adrianne , Hatton Charlie , Bryant Barbara M. , Sims Robert J.
Published: August 2014

The SWI/SNF family of chromatin remodeling complexes is critical for the mobilization of nucleosomes during transcription, DNA replication and repair, and cell division. SWI/SNF consists of a central ATPase subunit (BRG1 or BRM) and 10-12 additional subunits whose identity lends specificity to the complex. Abundant genomic data indicates that SWI/SNF components are frequent targets of homozygous loss of function mutations in a variety of tumor types. These loss of function mutations alter the composition and activity of the SWI/SNF complex, resulting in a tumor-specific chromatin state. In this work we describe a strategy for targeting this tumor-specific chromatin state using synthetic lethal RNAi screening. We have assembled and validated a focused shRNA library targeting 120 factors that act on chromatin, including histone methyltransferases, histone demethylases, and bromodomain-containing proteins. The Constellation shRNA library was transduced into several cell lines containing mutations in SWI/SNF components in addition to a panel of reference cell lines in order to identify factors that are specifically essential in those cell lines with altered SWI/SNF activity. Primary screening generated a list of genes that are potentially synthetically lethal with loss of function mutations in SWI/SNF components. Experiments are underway to confirm these genetic relationships and to obtain mechanistic insight into the role of these relationships in the context of chromatin biology. Most importantly, we will evaluate these genes as novel therapeutic targets in cancers with altered SWI/SNF function.

This abstract is also presented as Poster A19.

Citation Format: Andrew R. Conery, Adrianne Neiss, Charlie Hatton, Barbara M. Bryant, Robert J. Sims, III. Targeting altered SWI/SNF function through synthetic lethal RNAi screening. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR02.
Abstract PR07: Synthetic lethal screens in zebrafish
Authors: Sidi Samuel
Published: August 2014

The zebrafish embryo enables high-content screens in the intact, whole vertebrate. The system is particularly amenable to drug screening (up to 96-well format), and the mechanism of action of identified compounds can be readily dissected via morpholino or mRNA microinjections in live embryos (Sidi et al., Cell 133:864-77, 2008). We are exploiting zebrafish lines bearing cancer-relevant mutations (e.g., p53, pten, brca2 mutants) as platforms for synthetic lethal screens, focusing mainly on libraries of kinase inhibitors or FDA-approved compounds. Drugs that selectively induce cell lethality in mutant but not wild-type embryos are rescreened against panels of genetically annotated human cancer cell lines. A key advantage of the whole-embryo approach is the ability to identify those cell- or tissue-types in which the synthetic interaction occurs, thus guiding our human cancer cell-based validation screens. We will discuss the results of our large-scale screens targeted to p53, performed in the presence or absence of ionizing radiation-induced DNA damage.

This abstract is also presented as Poster A11.

Citation Format: Samuel Sidi. Synthetic lethal screens in zebrafish. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR07.
Abstract PR08: Identifying effectors of WT1 that regulate KRAS-induced senescence and chemoresistance in non-small cell lung cancer
Authors: Chen Ron , Simpson David R. , Zheng Yanyan , Vaka Dedeepya , Vicent Silvestre , Sweet-Cordero Alejandro
Published: August 2014

Activating KRAS mutations occur frequently in non-small cell lung cancer (NSCLC), and are associated with poor response to chemotherapy and overall survival. We previously used an oncogenic KRAS gene expression signature as the basis for a negative selection screen for synthetic lethal interactions with mutant KRAS. We identified WT1 as a transcription factor that bypasses oncogene-induced senescence caused by KRAS signaling. Based upon the success of this approach, we derived an improved oncogenic KRAS signature using a novel strategy that compares gene expression in sorted primary mouse tumor cells to human NSCLC gene expression datasets to predict transcription factors that are potential effectors of KRAS. Based on these bioinformatic predictions, we conducted a pooled shRNA negative selection screen in a panel of KRAS mutant and wild-type NSCLC cell lines, which validated known mutant KRAS synthetically lethal partners such as WT1 and TBK1, and identified novel candidates. We are evaluating these hits for their ability to regulate the KRAS gene expression signature and facilitate KRAS-driven tumorigenesis. In addition to its ability to bypass KRAS-induced senescence, we have shown that WT1 expression increases resistance to chemotherapy in non-small cell lung cancer cells. Using RNA-Seq to analyze differential gene expression and alternative splicing of transcripts, we have further identified key pathways and targets downstream of WT1 signaling that could play a role in regulating senescence and chemosensitivity in KRAS-driven tumors. We are using the results from these studies to define downstream effectors of WT1 in KRAS driven tumors that can be targeted to induce senescence and improve response to chemotherapy in NSCLC.

This abstract is also presented as Poster A29.

Citation Format: Ron Chen, David R. Simpson, Yanyan Zheng, Dedeepya Vaka, Silvestre Vicent, Alejandro Sweet-Cordero. Identifying effectors of WT1 that regulate KRAS-induced senescence and chemoresistance in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR08.
Abstract A125: Universal homogenous bioluminescent assay to monitor the effect of modulators on various classes of methyltransferases in vitro.
Authors: Goueli Said A. , Hsaio Kevin
Published: August 2014

Methylation/demethylation of DNA and proteins play major roles in modulation of the epigenome and has been implicated in a wide variety of human diseases. Recent biochemical and biological data suggest that the enzymatic activities of several of these enzymes have pathogenic roles in cancer, inflammation, and neurodegenerative diseases. Thus, pharmacological modulation of these enzymes by small molecules will be beneficial in developing novel therapeutics for multiple unmet medical needs. Towards this goal of searching for activators/inhibitors of these enzymes for the development of next generation of drugs, screening assays for these modulators are urgently needed. To address these unmet needs, we have developed a novel assay that monitors the activities of these enzymes and their modulation by small molecules. The assay is bioluminescent based, HTS formatted, and highly sensitive. The assay is universal since it is based on monitoring the formation of the universal product S-adenosylhomocysteine (SAH), i.e., capable of detecting changes in activity of a broad range of methyltransferases such as DNA, protein, and small molecules methyltransferases. In addition, the assay has been validated for all classes of protein methyltransferases (lysine and arginine), and with different types of substrates (small peptides, large proteins, or even nucleosomes). This enables determining the specificity of these enzymes and their substrate requirements. The assay has high signal to background and low C.V. The assay is robust (Z’ value>0.7) and has been validated using various plate densities such as 96-, 384, and 1536-well plates. A strong feature of this assay is its utility with broad range of substrates with no limitations on the use of high concentrations of substrates or the composition of the substrates (short vs. long peptides), thus enabling the generation of kinetic data and determining the mechanism of action of various modulators of methyltransferases of interest.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A125.

Citation Format: Said A. Goueli, Kevin Hsaio. Universal homogenous bioluminescent assay to monitor the effect of modulators on various classes of methyltransferases in vitro. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A125.
Abstract A126: Change in status promoter of 5 panel gene during progression of atypical hyperplasia to breast cancer.
Authors: Joaquina Martínez-Galán , Delgado Juan Ramon , Torres-Torres Blanca , Del Moral Rosario , Ríos Sandra , Núñez M. Isabel
Published: August 2014

Background: DNA methylation is the main epigenetic modification in cancer. The pathological features of breast cancer follow a sequential progression from transition of a normal cell to benign proliferative hyperplasia, hyperplasia with atypia, in situ carcinoma and, eventually, to invasive and metastatic disease. Purpose: This study was designed to evaluate the changes in promoter CpG islands hypermethylation during breast cancer progression from pre-invasive lesions to invasive ductal carcinoma and different methylation patron with benign breast disease.

Material and Methods: We performed analysis for the methylation status of 5 promoter CpG island (ESR1, E-Cad, APC, RARB, and 14-3-3-; promoters) in a group with women with normal breast tissue n=74; other group with epithelial atypia/ atypical ductal hyperplasia (n=20), ductal carcinoma in situ (DCIS, n=14) and invasive ductal carcinoma (IDC, n=107).

Results: Relative serum levels of methylated gene promoters ESR1 and 14-3-3-; showed significant differences between IDC y normal breast tissue (were significantly higher in IDC than normal breast tissue) and between normal breast tissue and epithelial atypia/ atypical ductal hyperplasia/ ductal carcinoma in situ. There are no differences between IDC and epithelial atypia/ atypical ductal hyperplasia/ ductal carcinoma in situ when 14-3-3-; and ESR1 mean values are compared. In fact, the present findings showed a frequent presence of methylated 14-3-3-; in sera from women with breast benign disease, which could be considered the result of a proliferative alteration of epithelial and stromal components of the breast. Which may indicate presence of occult benign breast disease, although other possible sources of this DNA include normal tissues, which show higher methylation values with increasing age; which might undergo lysis during tumor growth; or premalignant lesion during tumor growth; or premalignant lesion.

Conclusions: These findings showed that promoter CpG island methylation changed significantly in pre-invasive lesions, and breast cancer, suggesting that CpG island methylation of tumor-related genes is an early event in breast cancer progression.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A126.

Citation Format: Martínez-Galán Joaquina, Juan Ramon Delgado, Blanca Torres-Torres, Rosario Del Moral, Sandra Ríos, M. Isabel Núñez. Change in status promoter of 5 panel gene during progression of atypical hyperplasia to breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A126.
Abstract A128: Zebularine, a DNA methyltransferase inhibitor, increases apoptosis and decreases cell proliferation, cell cycle arrest and clonogenic capacity on childhood medulloblastoma cell lines.
Authors: Andrade Augusto F. , Borges Kleiton S. , Suazo Veridiana K. , Castro-Gamero Angel Maurício , Queiroz Rosane G. P. , Scrideli Carlos A. , Tone Luiz Gonzaga
Published: August 2014

Medulloblastoma (MB) is the most common malignant brain tumors in childhood, accounting for 20 % of all primary pediatric intracranial tumors. Although conventional therapies can cure a large proportion of patients with medulloblastoma, the majority of survivors suffer from long-term side effects, including developmental and neurological deficits. Thus, new approaches and therapeutic drugs are needed. Epigenetic drugs such as inhibitors of DNA methyltransferases (iDNMTs) have shown antineoplastic effects in different tumors. Zebularine (ZB) is a potent inhibitor of DNA methylation and has been associated with induction of apoptosis and enhancing tumor chemo- and radiosensitivity. However, its effects on childhood MB have not been previously reported. Herein, this study shows the preliminary data from the effects of ZB on cell proliferation, cell cycle, clonogenic capacity and apoptosis of childhood MB cells.

DAOY, ONS-76, UW-402 and UW-473 MB cell lines were used in all functional studies. Cell proliferation by XTT® assay, clonogenic capacity, cell cycle and apoptosis by flow cytometry using propidium iodide and anexin-V staining were performed using different doses of ZB (50-800µM). Statistical analysis was performed by one or two-way ANOVA and Bonferoni post-hoc.

ZB decreased cell proliferation and clonogenic capacity in all cell lines in a time- and dose-dependent manner, respectively (P< 0.05). ZB also caused apoptotic cell death in MB cells, where the percentage of apoptotic cells significantly increased after treatment compared control (P<0.05), reaching 60% on DAOY cells. Since the dose of 200uM, the drug showed a cell cycle arrest in S-phase in UW-473 and UW-402 cell lines and in G2/M phase in ONS-76 and DAOY cells after 72h of exposure.

These results indicate that ZB may be a promising drug for MB treatment and it confirms the potential of demethylating drugs in cancer treatment. Others studies and novel assays are being conducted to better understand these findings.

Financial Support: FAPESP (process no. 2011/22440-7)

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A128.

Citation Format: Augusto F. Andrade, Kleiton S. Borges, Veridiana K. Suazo, Angel Maurício Castro-Gamero, Rosane G. P. Queiroz, Carlos A. Scrideli, Luiz Gonzaga Tone. Zebularine, a DNA methyltransferase inhibitor, increases apoptosis and decreases cell proliferation, cell cycle arrest and clonogenic capacity on childhood medulloblastoma cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A128.
Abstract A134: Functional perturbation of PARP6 affects centromeric proteins and is associated with increased multi-polar spindles in breast cancer cell lines.
Authors: GROSSKURTH SHAUN , Petteruti Philip , Wang Xin , Mikule Keith
Published: August 2014

Poly(ADP-ribose) polymerases (PARP) are a family of 17 enzymes that catalyze the transfer of the ADP-ribose from NADH to post-translationally modify (PTM) acceptor proteins. Currently, the PTM regulation of proteins by PARPs have been demonstrated to regulate numerous signaling cascades including but not limited to DNA damage response and tumor development for PARP1-3 as well as telomere maintenance, spindle assembly, vesicular movement, and regulation of beta-catenin destruction complex for PARP5a-5b. Although progress on the biological role and therapeutic potential for some PARPs has been made, the function of and possible therapeutic application for other PARPs are not fully understood. Using cell free enzyme and cell-base multi-polar spindle assays, semi-selective PARP6 inhibitors AZ482 and AZ108 were identified. From in vitro PARP6 knock-down and AZ108 pharmacological studies in breast cancer cell lines, PARP6 perturbation was demonstrated to disrupt spindle pole clustering. To identify PARP6 protein substrate that may contribute to the spindle pole clustering defect, an in vitro acceptor protein substrate screen was performed using high-density protein microarrays containing more than 8,000 unique proteins. From this screen, an enrichment for centromeric and microtubule organizing proteins was identified. Of these centromeric PARP6 protein substrate, CHEK1 and CENP were shown to be modulated by AZ108 treatment in breast cancer cell lines. These results implicate PARP6 in a role for stablization of the spindle assembly during mitosis.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A134.

Citation Format: SHAUN GROSSKURTH, Philip Petteruti, Xin Wang, Keith Mikule. Functional perturbation of PARP6 affects centromeric proteins and is associated with increased multi-polar spindles in breast cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A134.
Abstract A136: Proteome analysis reveals candidate genes regulated by BLID, a novel drug-inducible apoptotic target, in breast cancer cells.
Authors: Yadavalli Sivaramakrishna , Eidelman Ofer , Srivastava Meera , Kasid Usha N.
Published: August 2014

BLID, BH-3 Like motif containing Inducer of cell Death, is a strong prognostic factor in invasive breast cancer (1). Frequent lack of BLID in breast cancer has been associated with triple-negative breast cancer (TNBC), African American ethnicity and younger women. Significant correlations exist between BLID negative breast cancer and declines in overall survival, local relapse-free survival and distant metastasis-free survival. In dose response and time course studies, BLID expression was found to be induced by chemotherapeutic drugs including doxorubicin, 5-fluorouracil and cisplatin in MCF-7 breast cancer cells. In addition, exogenous expression of BLID cDNA nanocomplex led to significant increase in chemosensitivity in SKBr3 (ER-ve/PR-ve) and MDA-MB231 (TNBC) breast cancer cell lines. Here we have investigated the effects of BLID knockdown on protein expression profiles in representative hormone responsive (MCF-7) and TNBC breast cancer cells (MDA-MB231) with a goal to identify a core translational component signifying BLID activity in breast cancer cells. BLID shRNA lentiviral particles were generated using pLKO.1-TRC containing BLID shRNA clone 47 according to the supplier's manual (Sigma-Aldrich). Breast cancer cells were stably transfected with BLID shRNA or scrambled shRNA. The pooled stable clones were tested for BLID expression by western blot analysis. Significant knockdown of BLID protein was achieved in breast cancer cells transfected with BLID shRNA (% inhibition of BLID expression vs. scrambled shRNA: MCF-7, 69%; MDA-MB231, 63%). The BLID proteome analysis was performed using antibody microarrays as we reported earlier (2). We have observed distinct and overlapping BLID-centric protein profiles in MCF-7 and MDA-MB231 cells. Among the top-ranked genes, BLID knockdown correlated with increased expression of PI3-kinase catalytic subunit α and DEK oncogene in MCF-7 cells, and increased expression of myeloid cell leukemia sequence 1 (MCL-1) in MDA-MB231 cells. Remarkably, BLID depletion correlated with decreased expression of tumor protein p73 and cAMP-dependent protein kinase (PKA Type Iα) in both cell lines. Thus, we have identified a working platform of genes signifying BLID expression in breast cancer cells. Validation of the top-ranked genes in various models of breast cancer is ongoing and data will be presented.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A136.

Citation Format: Sivaramakrishna Yadavalli, Ofer Eidelman, Meera Srivastava, Usha N. Kasid. Proteome analysis reveals candidate genes regulated by BLID, a novel drug-inducible apoptotic target, in breast cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A136.
Abstract A146: Preclinical models relevant to the treatment of post-menopausal breast cancer.
Authors: Pazolli Ermira , Mazzola Anne Marie , Curwen Jon , Weir Hazel , Kiessel Casey , Grondine Michael , D'Cruz Celina , Zinda Mike
Published: August 2014

The past decades have witnessed immense progress in the treatment of hormone-sensitive breast cancer. However, despite the success of agents targeting estrogen activity, a significant proportion of patients will experience relapse due to endocrine therapy resistance. In order to better understand the underlying causes and enable successful targeting of implicated pathways, preclinical models that recapitulate the post-menopausal setting are essential. To this end, we have developed a panel of estrogen receptor (ER+) models with varying senstivity to endocrine therapies. Our panel includes models of long-term estrogen-deprivation (LTED), a surrogate for longterm exposure and relapse on aromatase inhibitors. Using both xenograft (MCF7LTED, HCC1428LTED) and primary tumor models (BR0555fLTED and CTC-174LTED), we have utilized them to benchmark standard of care treatments and test the efficacy of novel agents. Importantly, we demonstrate that these models closely mimick the post-menopausal hormonal millieu found in patients and recapitulate pharmacodynamic responses observed in the clinic. As such, these are instrumental tools to uncovering endocrine resistance mechanisms and provide a potentially predictive platform for clinical efficacy.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A146.

Citation Format: Ermira Pazolli, Anne Marie Mazzola, Jon Curwen, Hazel Weir, Casey Kiessel, Michael Grondine, Celina D'Cruz, Mike Zinda. Preclinical models relevant to the treatment of post-menopausal breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A146.
Abstract A151: Identification of antibody fragments specific for high-grade prostatic intraepithelial neoplasia cells via SELEX.
Authors: Hajiran Cyrus J. , Sooter Letha J.
Published: August 2014

The purpose of this study is to develop a minimally invasive molecular tool for the early detection and specific treatment of prostate cancer. A human non-immune single-chain Fragment variable (scFv) library with 10∧9 diversity has been displayed by a yeast host surface with the ability to bind to whole cell targets. This protein library was cloned into plasmids and transformed into the yeast strain Saccharomyces cerevisiae. Surface expression of the scFv library was achieved by expressing the scFv under control of a galactose promoter preceding incubation with the target cell line.

A molecular recognition element (MRE) specific for HGPIN cells will be selected for through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). From an initial scFv library with 10∧9 diversity, seven rounds of selection will be completed in order to enrich the antibody fragment library that binds only to the prostate cancer cell line HGPIN and not those that bind to normal and cancerous cells of the prostate epithelium. This involves screening a library of random molecules for their ability to bind to a target; non-binding molecules are removed and binding molecules are amplified. Amplified molecules are subjected to negative targets, and molecules that don't bind to these are amplified, thus completing a round of selection. Stringency is increased through each round of selection by shortening the yeast + prostate cell incubation period and decreasing the number of target cells used in incubation. After multiple rounds, one or a few strong binding, highly specific molecules are selected for.

Diversity of the library is decreased with each round of selection. This is followed by amplification of the remaining yeast. Sequencing is performed throughout the selection process in order to monitor both the decrease in diversity and the emergence of the most selective sequences. The obtained MRE will be useful for specific diagnosis in the pre-malignant stages of prostate cancer. It may also be conjugated to drugs for targeted therapeutic applications.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A151.

Citation Format: Cyrus J. Hajiran, Letha J. Sooter. Identification of antibody fragments specific for high-grade prostatic intraepithelial neoplasia cells via SELEX. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A151.