bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B2-35: Efficient exploration of multi-cancer networks by generalized covariance selection and interactive web content
Authors: Kling Teresia , Johansson Patrik , Sanchez Jose , Marinescu Voichita D. , Jornsten Rebecka , Nelander Sven
Published: May 2016

Statistical network modeling techniques are increasingly important tools to analyze cancer genomics data. However, current tools for network construction and interpretation are not designed to work across multiple diagnoses and technical platforms, thus limiting their applicability to comprehensive pan-cancer datasets such as the Cancer Genome Atlas (TCGA). Here, we describe a novel strategy to construct and analyze integrative network models heterogeneous data from multiple cancers. First, we introduce a generalization of sparse inverse covariance selection (SICS) designed to integrate genetic, epigenetic and transcriptional data from multiple cancers into a comparative network. The algorithm is shown to be statistically robust, effective at detecting direct pathway links in data from The Cancer Genome Atlas (TCGA), and uses a new strategy involving non-informative priors to balance different cancers and data types. Second, we propose to rationalize the interpretation of the derived networks by a new and publicly accessible tool (cancerlandscapes.org), in which derived models are explored as interactive web content, linked to several pathway and pharmacological databases. To evaluate the performance of the method, we constructed a model of genetic, epigenetic and transcriptional data for eight TCGA cancers, using data from 3900 patients. The derived model rediscovered known mechanisms and contained interesting predictions. Possible applications include the prediction of regulatory relationships between genes in particular cancers, comparison of network modules in across multiple forms of cancer, and identification of drug targets in relation to network structure.

Citation Format: Teresia Kling, Patrik Johansson, Jose Sanchez, Voichita D. Marinescu, Rebecka Jornsten, Sven Nelander. Efficient exploration of multi-cancer networks by generalized covariance selection and interactive web content. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-35.
Abstract IA03: Towards the next clinical option: Experience from a precision cancer medicine trial
Authors: Rubin Mark A.
Published: May 2016

Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low frequency mutations. We describe an evidence-based precision medicine program designed to bring whole exome sequencing (WES) into clinical practice and illustrate how this can be utilized to identify novel predictive biomarkers associated with exceptional systemic response.

Metastatic and treatment-resistant cancer patients are prospectively enrolled for paired metastatic tumor and normal WES. Using a comprehensive computational pipeline, point mutations, indels and copy number alterations are detected. Mutations are categorized as Category I-III based on actionability and a report is generated and discussed in tumor board. Patients are followed prospectively for correlation of molecular information with clinical response and patient outcomes. A rapid response functional team follows on key findings to expand actionability based on the WES results.

In the first year, 154 tumor-normal pairs from 97 patients were sequenced, with an average coverage of 95X of over 21,000 genes and 16 somatic alterations detected on average per patient. In total, 16 mutations were Category I, 98 were Category II, and 1474 were Category III. Tumor purity ranged from 14 to near 100%. Among unexpected findings, a prostate cancer exceptional responder was identified that harbored a somatic hemizygous deletion of the DNA repair gene FANCA and likely loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response.

The establishment of a clinical program for whole exome sequencing of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response.

Citation Format: Mark A. Rubin. Towards the next clinical option: Experience from a precision cancer medicine trial. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr IA03.
Abstract IA14: Genomic alterations dysregulate cancer genes by modulating microRNA activity
Authors: Chiu Hua-Sheng , Yang Xuerui , Califano Andrea , Sumazin Pavel
Published: May 2016

microRNAs play key roles in cancer etiology, and recent evidence suggests that targets of the same microRNA programs are stoichiometrically coupled via a competitive endogenous RNA (ceRNA) mechanism. Consequently, aberrant expression of multiple targets of the same microRNA program dysregulate coupled ceRNA- oncogenes and tumor suppressors.

We computationally inferred and experimentally validated that coordinated copy number and methylation alterations contribute to physiologically significant in-trans dysregulation of hundreds of coupled oncogenes and tumor suppressors in multiple tumor contexts. Both high-throughput perturbation assays and low-throughput mutational assays confirmed significant ceRNA-mediated regulation of cancer genes in eight tumor contexts, including ESR1 and APC in breast and colon cancer, respectively.

Our analysis infers roles for previously uncharacterized genomic alterations and it suggests that ceRNA-mediated interactions account for a substantial fraction of cancer's missing genomic variability.

Citation Format: Hua-Sheng Chiu, Xuerui Yang, Andrea Califano, Pavel Sumazin. Genomic alterations dysregulate cancer genes by modulating microRNA activity. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr IA14.
Abstract IA20: Post-transcriptional regulation of metastatic progression
Authors: Tavazoie Sohail F.
Published: May 2016

Post-transcriptional regulation represents an increasingly studied layer of gene expression control. In recent years, it has become increasingly evident that deregulation in post-transcriptional control mechanisms represents an important feature of the aggressive malignant phenotype. We have found that distinct cancers arising from various tissues such as the breast, skin, and the colon modulate unique tissue-specific sets of small non-coding RNAs (microRNAs) as they acquire a highly metastatic phenotype. We have used these microRNAs as molecule probes to better understand the molecular and cellular biology underlying metastatic progression. These studies have revealed surprising pathways and mechanisms utilized by cancer cells to colonize metastatic microenvironments. We find that while distinct tissues such as breast and melanoma might modulate distinct miRNAs and downstream target genes, the cellular phenotypes impacted by these pathways en route to metastatic colonization can be similar. Importantly, clinical association studies using human specimens support the roles for these miRNAs and their downstream effectors in metastatic progression. These basic studies into the molecular and cellular biology of metastatic progression have revealed key target genes whose pharmacological modulation in proof-of-concept pre-clinical studies have revealed robust therapeutic effects.

Citation Format: Sohail F. Tavazoie. Post-transcriptional regulation of metastatic progression. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr IA20.
Abstract A03: Inhibition of the histone/protein acetyltransferase, Tip60, impairs Foxp3 acetylation and Treg function and thereby promotes antitumor immunity
Authors: Hancock Wayne W. , Wang Liqing , Newick Kheng , Beeler Aaron , Albelda Steven M.
Published: May 2016

Although the important role of Foxp3+ Tregs in suppressing immune responses to solid tumors is well accepted, to date, there has been little success in targeting this population other than by their transient depletion or “secondary” modulation by mAbs to CTLA-4. In contrast, we have undertaken a mechanism-driven approach to Treg targeting based on our ongoing research into the post-translational modifications that regulate Foxp3, such as its acetylation by one or more histone/protein acetyltransferase (HAT) enzymes. Our analysis of each of the main HAT families has shown that members of the Myst family have the greatest contribution, with Tip60 deletion leading to lethal autoimmunity within 3 weeks of birth. We have now translated these insights into the development and testing of a new pharmacologic inhibitor of Tip60 (Tip60i) that binds to the Ac-CoA site of Tip60. First, submicromolar levels of this Tip60i impaired Treg function in vitro but preserved conventional T cell proliferation. Second, Tip60 administration in vivo (10 mg/kg/d, 14 d) revoked Treg-dependent allograft tolerance induced by CD40L mAb plus donor splenocyte transfusion, and restored T cell-dependent cardiac allograft rejection. These studies illustrate the ability of the Tip60i to impair Treg functions while preserving protective effector T cell functions. Third, treatment of immunocompetent mice bearing established TC1 lung adenocarcinomas (which we have shown is a Treg-sensitive tumor) with Tip60i (1 mg/kg/d) significantly impaired tumor growth (p<0.001). While further medicinal chemistry of this compound is underway, including modification with phthalimide tags, our data provide the crucial first evidence that Tip60 is a druggable and potent target for modulation of Foxp3+ Treg function in vitro and in vivo, and show that Tip60 inhibition can enhance host anti-tumor immunity.

Citation Format: Wayne W. Hancock, Liqing Wang, Kheng Newick, Aaron Beeler, Steven M. Albelda. Inhibition of the histone/protein acetyltransferase, Tip60, impairs Foxp3 acetylation and Treg function and thereby promotes antitumor immunity. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A03.
Abstract A16: The crosstalk between Indoleamine 2,3 Dioxygenase (IDO) and cancer stem cells in murine melanoma: A potential modality of local control
Authors: Kocherla Marsha , Christianson Blake , Walker James , Qin Xu , Yu Jack C. , Baban Babak
Published: May 2016

Introduction: Indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme of tryptophan catabolism, has been strongly associated with the progression of malignancy and poor survival in melanoma patients. As a result, IDO is a leading target for interventions aimed at restoring melanoma immune surveillance. IDO, which is produced by cells including dendritic cells, macrophages, stem cells, and most tumor cells, potently suppresses immune responses by depleting local tryptophan and allowing the accumulation of tryptophan metabolites. However, the actual cellular and molecular mechanisms underlying the immunoregulatory function of IDO remain largely unknown. The relatively newly described cancer stem cells (CSCs) are tumor-initiating cells (TICs) that often survive therapy and give rise to second-line tumors by self-renewal and differentiation into multiple cell types. CSCs are proposed to persist in tumors as a distinct population and to cause relapse and metastasis by giving rise to new tumors. Therefore, development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients, especially those with metastatic disease. Methods: C57/BL6 mice were injected with B16F10 murine melanoma cell line (1 x 106 cells/mouse, subcutaneous). Tumor growth monitored. At 3mm, tumors were injected with an IDO Inhibitor (1 MT) (Group 1) twice weekly (20 mg/mouse per injection). Controls (Group 2) received vehicle only (PBS), Group 3 weekly Cisplatin (5mg/kg), and Group 4 both Cisplatin and 1MT (IDO inhibitor). Once the first tumor reached 12mm in size, the mice were sacrificed and tumor cells subjected to flow-cytometry, and statistical analysis, and p-values <0.05 considered significant. Results: Our data showed for the first time that IDO was expressed by CSCs in a murine melanoma model. These IDO-expressing CSCs may be capable of suppressing T lymphocyte infiltration into the tumor microenvironment, which was reversed by the IDO inhibitor, 1-methyl-tryptophan. Conclusion: Our findings offer an important new line of evidence that interventional targeting of IDO activity could be used to restore tumor immunity by relieving IDO-mediated immune suppression of CSCs in the tumor microenvironment as well as in tumor cells themselves.

Citation Format: Marsha Kocherla, Blake Christianson, James Walker, Xu Qin, Jack C. Yu, Babak Baban. The crosstalk between Indoleamine 2,3 Dioxygenase (IDO) and cancer stem cells in murine melanoma: A potential modality of local control. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A16.
Abstract A17: The CEBPD transcription factor: A signaling hub for promotion of cancer cell stemness
Authors: Kuppusamy Balamurugan , Mendoza-Villaneuva Daniel , Summers Glenn , Sharan Shikha , Sterneck Esta
Published: May 2016

Our previous studies showed that the transcription factor CCAAT/enhancer binding protein delta (CEBPD) promotes tumor metastasis in a mouse model of mammary tumorigenesis (MMTV-Neu) (Balamurugan et al., EMBO J, 2010). Here we show that CEBPD serves as a signaling hub, which integrates and amplifies signaling pathways that promote stemness in breast cancer and glioblastoma as well as in embryonic carcinoma stem cells. This work identifies a mechanism by which hypoxia and inflammation promote cancer stem-like cells (CSCs), which are implicated in tumor progression, metastasis, and therapy resistance.

We have shown in our earlier studies that CEBPD promotes inflammatory signaling and hypoxia adaptation by inhibiting the expression of the F-box protein FBXW7, a bona fide tumor suppressor (Balamurugan et al., EMBO J 2010; Nature Communications 2013). Because FBXW7 can target the stem cell factor Notch for degradation, we hypothesized that CEBPD can promote cell stemness. We tested this hypothesis using a variety of established breast and glioblastoma tumor cell lines/xenografts, primary mouse mammary tumor cells and tissues, recent patient-derived glioblastoma cell lines, as well as embryonic stem/cancer cell lines. Deletion or depletion of CEBPD reduced the number of stem cells as determined by surface markers, sphere formation, limiting dilution xenografts, and by expression of stemness genes. Mechanistic studies in culture models show that CEBPD is expressed in CSCs and connects and amplifies both hypoxia (HIF-1) and inflammation (IL-6/STAT3) pathways to increase Notch1 activity and cell stemness. Furthermore, we found that CEBPD also directly activates the expression of stemness associated genes such as OCT4, Sox2, KLF4, Myc, Nanog, CD44 and CD133. Taken together, this study provides novel insights into the molecular mechanisms that promote cancer cell stemness, and identifies a central role for CEBPD in integrating HIF-1α,

IL-6/STAT3, and Notch1 signaling, while also activating expression of stemness factors. These results strongly suggest that pharmacological inhibition of CEBPD signaling may effectively target CSCs.

Citation Format: Balamurugan Kuppusamy, Daniel Mendoza-Villaneuva, Glenn Summers, Shikha Sharan, Esta Sterneck. The CEBPD transcription factor: A signaling hub for promotion of cancer cell stemness. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A17.
Abstract A18: The tankyrase inhibitor G007-LK inhibits small intestine Lgr5+ stem cell proliferation without altering tissue morphology
Authors: Norum Jens Henrik , Skarpen Ellen , Brech Andreas , Kuiper Raoul , Waaler Jo , Krauss Stefan , Sørlie Therese
Published: May 2016

Tankyrase enzymes play crucial roles in the Wnt signaling pathway, involved in the regulation of intestinal stem cells and tissue homeostasis. Aberrant Wnt signaling is known to cause intestinal cancers and the tankyrase inhibitor G007-LK has previously been shown to inhibit tumor growth in an APC-mutant colorectal cancer xenograft model.

We have used in vivo models to address the effect of G007-LK on small intestine tissue homeostasis. The mice were treated for up to 3 weeks with the tankyrase inhibitor, without affecting the body mass or observed clinical condition of the mice. H&E staining of fixed small intestine tissue sections showed no significant differences in the general tissue morphology between control and G007-LK treated mice. In the presence of G007-LK, lineage tracing from the Lgr5+ intestinal stem cells was significantly reduced compared to control mice. When the G007-LK treatment was stopped two days prior to initiation of lineage tracing, there were no significant difference in the number of traced cells between the mice pretreated with G007-LK and untreated control group. Immunohistochemistry (IHC) staining for the proliferation marker Ki67 showed a slight reduction in the number of positive cells in the small intestine of the G007-LK treated compared to control mice. As expected, in the crypt base cells there was a reduction of nuclei staining positive for β-catenin in the G007-LK treated compared to control mice, suggesting repressed Wnt/β-catenin signaling.

Taken together, our data show that the tankyrase inhibitor G007-LK is well tolerated by the mice. The tankyrase inhibitor reduced the proliferative activity of the small intestine Lgr5+ stem cells, without altering the general morphology or function of the tissue.

Citation Format: Jens Henrik Norum, Ellen Skarpen, Andreas Brech, Raoul Kuiper, Jo Waaler, Stefan Krauss, Therese Sørlie. The tankyrase inhibitor G007-LK inhibits small intestine Lgr5+ stem cell proliferation without altering tissue morphology. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A18.
Abstract A27: An in vivo gain-of-function screening identified the Williams-Beuren syndrome gene GTF2IRD1 as a mammary tumor promoter
Authors: Huo Yongliang , Su Yinghao , Cai Qiuyin , Macara Ian
Published: May 2016

Precision medicine has shown great efficacy in oncology, but its broad application requires a full understanding of the cancer genome. We have performed the first gain-of-function screening in mice to functionally identify candidates from a “druggable” gene set for their roles in breast cancer. We transduced mammary gland stem/progenitor cells isolated from MMTV-ErbB2 transgenic mice with a lentiviral cDNA library and implanted them into the cleared fat pads of syngeneic recipients. The transplanted mammary gland cells formed a normal mammary ductal tree within 6 weeks. After another 8 months latency, small lesions were detected along the mammary ducts in mice harboring cells that had been transduced with the lentiviral library. We isolated genomic DNA from these lesions to amplify any lentiviral cDNA sequences within the lesions and identified individual genes by sequencing. Gain-of-function of five genes, including RET, GTF2IRD1, ADORA1, LARS2, and DPP8, was found to promote mammary tumor growth consistently. We further studied one novel tumor promoting gene, transcription factor GTF2IRD1. GTF2IRD1 regulates the expression of multiple genes in the mammary gland system, including OPN, TβR2, and BMPR1b. In tissue culture, high levels of GTF2IRD1 inhibited cell proliferation but increased the colony formation ability of the un-transformed mammary gland cell line MCF10A. In mice, the mis-regulation of GTF2IRD1, OPN, TβR2, and BMPR1b, also individually promoted mammary cancer development. Silencing of TβR2 delayed the incidence of tumor formation by GTF2IRD1. We also found that GTF2IRD1 is highly expressed in human breast tumors, correlating with high tumor grades and poor prognosis.

In conclusion, we performed the first gain-of-function screening in vivo for breast cancer promoting genes. All candidate genes from this study are potential drug targets to treat cancer. We showed that GTF2IRD1 promotes breast cancer development in mice through regulation of its downstream targets, and correlates clinically with high tumor grades and poor prognosis. We believe our in vivo screening method is readily expandable to whole genome annotation of cancer genes.

Citation Format: Yongliang Huo, Yinghao Su, Qiuyin Cai, Ian Macara. An in vivo gain-of-function screening identified the Williams-Beuren syndrome gene GTF2IRD1 as a mammary tumor promoter. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A27.
Abstract A46: Cellular apoptosis susceptibility (CAS) regulates integrin-beta1 and is required for tumor cell migration and invasion in hepatocellular carcinoma (HCC)
Authors: Winkler Juliane , Sticht Carsten , DiGuilio Amanda , Eiteneuer Eva Maria , Holzer Kerstin , Rössler Stephanie , Gretz Norbert , Breuhahn Kai , Schirmacher Peter , Ori Alessandro , Singer Stephan
Published: May 2016

Nuclear transport is an essential, highly selective process in eukaryotic cells which guarantees the bidirectional communication between the nucleus and the cytoplasm. Emerging evidence suggests that aberrant expression of nuclear transport factors may contribute to cancer formation and progression. We could recently show that the nuclear exporter Cellular apoptosis susceptibility (CAS) is overexpressed in hepatocellular carcinoma (HCC), the functional implications of which remain, however, not completely understood. Here, we integrated proteomics, transcriptomics and functional assays with patient data to further characterize the role of CAS in HCC. We found that CAS depletion in HCC cells led to deregulation of integrins, particularly integrin-beta1, and resulted in reduced cell migration and invasion. Moreover, we determined that high expression levels of CAS in HCC cases were associated with macroangioinvasion and poorer patient outcome. Our data indicate that CAS is linked to integrin signaling and correlates with an aggressive HCC phenotype.

Citation Format: Juliane Winkler, Carsten Sticht, Amanda DiGuilio, Eva Maria Eiteneuer, Kerstin Holzer, Stephanie Rössler, Norbert Gretz, Kai Breuhahn, Peter Schirmacher, Alessandro Ori, Stephan Singer. Cellular apoptosis susceptibility (CAS) regulates integrin-beta1 and is required for tumor cell migration and invasion in hepatocellular carcinoma (HCC). [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A46.
Abstract B05: Invadopodium formation is enriched in perivascular niches and leads to cancer cell intravasation
Authors: Bergman Aviv , Condeelis John , Gligorijevic Bojana
Published: May 2016

Invadopodia are actin-rich protrusions used for cancer cell movement across obstacles. In the past, they were mostly studied in cell cultures, where they form in contact with extracellular matrix. Our past work has suggested that elimination of key molecular components of invadopodia in cancer cells can lead to elimination of metastasis.

In the current study, we use high resolution multiphoton microscopy to understand how the tumor microenvironment controls formation of invadopodia, followed by cancer cell locomotion, intravasation and metastasis. We demonstrate that the motile cancer cells in breast carcinoma in vivo can exhibit either invadopodium-related, slow locomotion or invadopodium-independent rapid locomotion along collagen fibers. These two phenotypes occur in spatially separate tumor regions, the location of which can be predicted from measurements of microenvironmental features. For example, cancer cells form invadopodia predominantly in perivascular niches, regions surrounding major blood vessels and characterized by cross-linked collagen fibers and dense macrophages. Invadopodia in vivo extend towards blood vessels and their density decreases with blood vessel distance.

We observe that only cells with invadopodia are capable of intravasation, while fast locomotion results in cell dispersal throughout tumor. Further, either invadopodium-specific TkS5 knock-down or short-term inhibition of metalloproteases eliminate invadopodia and stop dissemination to lung. Interestingly, external modulation of collagen cross-linking also inhibits invadopodia, switching the cancer cell phenotype to fast locomotion.

Our results suggest that the perivascular niche is the site of invadopodium formation and cancer cell intravasation. Moreover, invadopodia and hence metastasis, can be inhibited by external modulation of the microenvironment. In summary, the presence of invadopodia is a great candidate for a novel early-metastasis marker, while their elimination via microenvironment modulation has therapeutic potential.

Citation Format: Aviv Bergman, John Condeelis, Bojana Gligorijevic. Invadopodium formation is enriched in perivascular niches and leads to cancer cell intravasation. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B05.
Abstract B17: Role of Sall2 transcription factor under genotoxic stress: A transcriptomic approach
Authors: Farkas Carlos A. , Fuentes Francisco , Rebolledo Boris , Makova Kateryna , Nekrutenko Anton , Castro Ariel , Pincheira Roxana
Published: May 2016

The induction of genotoxic stress by chemotherapy is one of the first line treatments to eliminate neoplastic cells through apoptosis or programmed cell death. Our laboratory has characterized the Sall2 transcription factor, conserved among vertebrates, as a stress-response molecule, and demonstrated both in vitro and in vivo its requirement in the cell death-response caused by genotoxic stress. Few transcriptional targets of Sall2 are known, thus, additional targets most likely remain to be identified. We isolated mouse embryonic fibroblasts (MEFs) from a murine model deficient in Sall2 and from its normal counterpart, and use them to characterize the Sall2-dependent transcriptional response upon genotoxic stress using RNA-seq experiments. Under control conditions we identified ninety-six Sall2-dependent genes involved in immune system, inflammation, cell adhesion and the response to wounding. Under genotoxic stress one hundred twenty six genes were differentially expressed and dependent on Sall2, these genes are mainly involved in immunity, development and cell-cycle regulation. Finally, we performed validation of the analysis using qPCR. With this experimental outline, we are expecting to gain insights on how Sall2 contributes to the transcriptional program under normal and genotoxic stress-induced conditions.

Citation Format: Carlos A. Farkas, Francisco Fuentes, Boris Rebolledo, Kateryna Makova, Anton Nekrutenko, Ariel Castro, Roxana Pincheira. Role of Sall2 transcription factor under genotoxic stress: A transcriptomic approach. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B17.
Abstract B18: A simple method to prepare oligonucleotide-conjugated antibodies for multiplex protein detection
Authors: Gong Haibiao , Holcomb Ilona , Ooi Aik , Wang Xiaohui , Majonis Daniel , Unger Marc , Ramakrishnan Ramesh
Published: May 2016

With rapid advances in nucleic acid detection technologies, genomics studies are being conducted in highly multiplexed formats. However, multiplex analysis is still a daunting task in the proteomics field; it would be of great interest to use genomics tools for protein detection. One way to accomplish this is to use oligonucleotide-conjugated antibodies to bind to specific protein targets, and to monitor conjugated-oligonucleotide levels rather than to measure protein levels directly. In this report we describe a simple method for preparing oligonucleotide-conjugated antibodies and discuss this method’s application in oligo extension reaction (OER) for multiplex protein detection. Conjugation is based on strain-promoted alkyne-azide cycloaddition (the Cu-free click reaction), in which the antibody is activated with a dibenzocyclooctyne (DBCO) moiety and linked covalently with an azide-modified oligonucleotide. The reaction condition and purification process were optimized to achieve maximum yield and best performance in the functional test. The OER assay employs a pair of antibody binders (two antibodies, each conjugated with its own oligonucleotide) developed for each protein target. The two oligonucleotides contain a six-base complementary region at their 3′ prime ends to allow annealing and extension by DNA synthesis enzymes to form a DNA template. The template is then detected by qPCR on the Biomark HD system. Distinct oligonucleotide sequences are assigned to different antibody binders to enable multiplex protein detection. When tested using recombinant proteins, some antibody binders, such as those specific to CSTB, MET, EpCAM and CASP3, had an estimated limit of detection (LOD) of about 0.06 pg/mL and a dynamic range of ≥5 logs. These antibody binders were used in multiplex protein detection in single cells using the C1™ system. The results in individual cells showed the expression of similar protein levels for CSTB and CASP3, but highly varied protein levels for EpCAM, CCNB1 and MUC1. This simple method for the preparation of oligonucleotide-conjugated antibodies is essential for the development of affordable multiplex protein assays, which has the potential to accelerate proteomics research.

Citation Format: Haibiao Gong, Ilona Holcomb, Aik Ooi, Xiaohui Wang, Daniel Majonis, Marc Unger, Ramesh Ramakrishnan. A simple method to prepare oligonucleotide-conjugated antibodies for multiplex protein detection. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B18.
Abstract B42: The sigma-2 receptor as a target for therapeutic drug delivery in triple negative breast cancer
Authors: Makvandi Mehran , Tilahun Estifanos D. , Lieberman Brian P. , Xu Kuiying , Zeng Chenbo , Hou Catherine , Mach Robert H.
Published: May 2016

Introduction: Triple Negative Breast Cancer (TNBC) is an aggressive form of breast cancer and typically results in poor patient prognosis. While other breast cancer subtypes benefit from targeted therapies, TNBC currently has no approved agents. The sigma-2 receptor has been validated as a proliferation marker and also a site for therapeutic drug delivery in ovarian and pancreatic cancer. In this study we evaluated the sigma-2 receptor as a target for therapeutic drug delivery in TNBC using a sigma-2 targeting vehicle conjugated to a small molecule activator of caspases(SMAC) mimetic, SW-IV-134.

Methods: Triple negative breast cancer cell lines; MDA-MB-231, HCC1937 and HCC1806 were tested for sensitivity to sigma-2 targeted compound SW-IV-134 vs. Taxol or the deconstructed conjugate subparts. 2 hr and 48 hr treatments we assessed to determine whether there was a significant in vitro targeting effect. Sigma-2 expression was quantified through radioligand saturation studies using cell homogenate tissue and the Ki value of SW-IV-134 for the sigma-2 receptor was calculated through competitive inhibition studies. Caspase 3/7 activation was assessed after 2 or 48 hr treatments.

Results: SW-IV-134 was the most potent treatment in MDA-MB-231 and HCC1937, while no difference from taxol was found in HCC1806. Sigma-2 receptor expression was found to be the highest in the MDA-MB-231 and no difference was found in HCC1937 and HCC1806. The Ki of SW-IV-134 for the sigma-2 receptor was 23 nM and is consistent with previously reported values. Caspase 3/7 activation was detected in all cell lines following treatment at 2 and 48 hr. The greatest caspase 3/7 activation was observed in the MDA-MB-231 cell line.

Conclusion: The sigma-2 receptor is expressed at different levels on the three TNBC cell lines and the receptor expression corresponds to therapeutic efficacy. SW-IV-134 was the most potent therapeutic tested in two of the three cell lines with no difference between taxol observed in HCC1806. This study shows the sigma-2 receptor is a target that can be exploited for therapeutic drug delivery in three TNBC cell lines and may provide a platform for future targeted therapy development for TNBC.

Citation Format: Mehran Makvandi, Estifanos D. Tilahun, Brian P. Lieberman, Kuiying Xu, Chenbo Zeng, Catherine Hou, Robert H. Mach. The sigma-2 receptor as a target for therapeutic drug delivery in triple negative breast cancer. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B42.
Abstract B44: Sulfated fucan inhibits tumor interaction with endothelial cells and tumor growth: Possible contribution of an antivascular endothelial growth factor (VEGF) neutralizing activity
Authors: Mignone Viviane , Figueiredo Camila Castro , Oliveira Aline , Kozlowski Eliene , Borsig Lubor , Pavão Mauro , Mourão Paulo , Morandi Veronica
Published: May 2016

The primary interaction between tumor and endothelial cells is mediated by endothelial selectins and sialyl-Lewis carbohydrates found on cancer cell surface. Heparin, a sulfated polysaccharide, binds to P- and L-selectins, an effect that has been associated with attenuation of metastasis. However, due to its strong anticoagulant activity, its use in anti-tumor therapy is restricted. Sulfated fucans from sea urchins have simple, unique structures of linear chains of sulfated α-L-fucose in well-defined repetitive patterns. In this study, we tested five sulfated fucans from different species of sea urchins for their ability to interfere with the tumor-endothelium interaction and compared our findings to those obtained with heparin. Our results show that sulfated fucan from L. variegatus (FucSulf I), bearing an unique 2,4-disulfated fucose residue and low anticoagulant activity, inhibits the adhesion of tumor cells to endothelium, tumor transendothelial migration and endothelial cell tubulogenesis, both by inhibiting P-selectin mediated tumor cell adhesion, and VEGF-dependent signaling in endothelial cells. FucSulf I also inhibited proliferation of several types of tumor cell lines in vitro and tumor growth in vivo. Our results indicate this sulfated fucan is a potential antitumor agent and a possible therapeutic alternative to heparin.

Citation Format: Viviane Mignone, Camila Castro Figueiredo, Aline Oliveira, Eliene Kozlowski, Lubor Borsig, Mauro Pavão, Paulo Mourão, Veronica Morandi. Sulfated fucan inhibits tumor interaction with endothelial cells and tumor growth: Possible contribution of an antivascular endothelial growth factor (VEGF) neutralizing activity. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B44.
Abstract IA01: Immune checkpoint blockade in cancer therapy: New insights, opportunities, and prospects for cures
Authors: Allison James P.
Published: May 2016

The existence of multiple non-redundant inhibitory pathways that limit T cell responses offers novel strategies for mobilizing the immune system to attack cancer cells. The best characterized of these immune checkpoints is CTLA-4, which inhibits T cell proliferation by interfering with the interaction of the costimulatory molecule CD28 with its ligands B7-1 and B7-2 on the surface of antigen presenting cells. Antibodies to CTLA-4 have proven effective against multiple tumor types in both pre-clinical and clinical studies. Ipilimumab, an antibody to human CTLA-4, showed long term (>4 years) survival benefit in about 20% of patients in a randomized, placebo-controlled trial in late stage melanoma. In 2011 it was approved by the FDA for treatment of late stage melanoma and is now a standard of care for that disease.

The mechanism(s) of action of anti-CTLA-4 are still being elucidated. We and others have shown that CLTA-4 limits T cell proliferation by a cell intrinsic mechanism. However, there is also evidence that anti-CTLA-4 has to engage the target on both effector (Teff) and regulatory (Treg) T cells. Thus anti-CTLA-4 exerts its anti-tumor effects by multiple mechanisms.

PD-1, another checkpoint, recruits a phosphatase and seems to interfere with T cell antigen receptor mediated signaling. It has two ligands, PD-L1 and PD-L2, which are both expressed on dendritic cells. However, many tumor cells also express PD-L1. Antibodies to PD-1 and PD-L1 have both shown objective responses against several tumor types in clinical trials with response rates of about 25%. A recent phase II trial of a combination of anti-PD-1 and anti-CTLA-4 in melanoma showed objective responses in about 50% of late stage melanoma patients.

Emerging data suggest that T cell responses are largely directed toward neoantigens arising as a result of mutational events associated with the carcinogenesis. While all tumors with antigens recognizable by the immune system should be targets for checkpoint blockade, it is becoming clear that certain types of tumors with lower burdens of mutations (eg. prostate, breast, and kidney cancer) present special challenges for immune therapy. Strategies for effective treatment of such tumors will be discussed.

Citation Format: James P. Allison. Immune checkpoint blockade in cancer therapy: New insights, opportunities, and prospects for cures. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA01.
Abstract IA03: From the clinic to the lab: Investigating immune responses to immune checkpoint therapies
Authors: Sharma Padmanee
Published: May 2016

Immune checkpoint therapies, including anti-CTLA-4, anti-PD-1 and anti-PD-L1, have led to significant clinical responses in cancer patients. To investigate immunologic changes and mechanistic pathways that are elicited by these therapies, we conducted pre-surgical clinical trials, which permit access to sufficient tumor tissues for laboratory studies. The first pre-surgical trial was conducted with anti-CTLA-4 (ipilimumab) in a cohort of patients with localized bladder cancer. This trial provided access to sufficient tumor-infiltrating lymphocytes to conduct phenotypic and functional studies on these cells, which indicated the ICOS/ICOSL pathway as relevant for anti-tumor immune responses in the setting of anti-CTLA-4 therapy. In addition, since standard agents that enable tumor cell death may allow for priming of a T cell immune response that can be augmented by combination with CTLA-4 blockade, we conducted a pre-surgical clinical trial with androgen deprivation (hormonal) therapy ADT+ anti-CTLA-4 (ipilimumab), in the setting of patients with regional, high-risk prostate cancer. These pre-surgical clinical trials, and other tissue-based clinical trials, led to the identification of biomarkers and additional targets. These data, which are being used to design future immunotherapy trials, will be discussed in greater details.

Citation Format: Padmanee Sharma. From the clinic to the lab: Investigating immune responses to immune checkpoint therapies. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA03.
Abstract IA17: Deciphering cancer genome evolution in heterogenous tumors
Authors: McGranahan Nicholas
Published: May 2016

Cancer drug resistance is almost inevitable in the majority of patients with advanced metastatic tumors. Intratumor heterogeneity, facilitating rapid tumor evolution, is one of the main causes of tumor adaptation and resistance to cancer therapies.

In this talk I will explore how cancer genome sequencing data can shed light on diversity within tumors and the processes shaping cancer genome evolution over space and time.

Harnessing both extensive multi-region and single-sequencing data we have investigated intra-tumor heterogeneity across 10 major cancer types, focusing on non-small cell lung cancer, clear cell renal cell carcinoma, and esophageal adenocarcinoma. I will discuss our findings shedding light on the extent to which mutations in known driver genes, and “actionable mutations,” linked to targeted therapies, are clonal or subclonal. Tumor phylogenies and whether patterns of tumor evolution can be deciphered in these cancer types will also be explored.

Temporal dissection of mutational signatures across cancer types will be used to reveal the dynamics of mutational processes during tumor evolution. I will explore how APOBEC mediated-mutagenesis can be linked to branched tumor evolution, and the acquisition of subclonal driver events across cancer types.

Finally, the challenges and clinical implications of intratumor heterogeneity will be discussed.

Citation Format: Nicholas McGranahan. Deciphering cancer genome evolution in heterogenous tumors. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA17.
Abstract IA25: Identifying and profiling tumor specific T cells using mass cytometry and highly multiplexed peptide-MHC tetramer staining
Authors: Newell Evan W.
Published: May 2016

Antigen specific T cells are critical initiators and orchestrators of the adaptive immune response. To better understand how T cells participate (appropriately or inappropriately) in the immune response to cancer, more meaningful categorizations of these cells are needed. For this purpose, a very large number of cellular parameters can be considered. Focusing on human CD8+ T cells, the utility of high dimensional mass cytometry (i.e., Cytometry by Time-Of-Flight, CyTOF) analysis will be discussed. By this approach, T cells can be probed with unprecedented detail by simultaneously evaluating surface marker expression, functional capacity and antigen-specificity. For antigen-specificity, this analysis can be performed in conjunction with a highly multiplexed method based on peptide-MHC tetramers, which allows simultaneous assessment of >500 different antigen specificities in a single sample. To visualize and understand the diversity of these cells, several computational data analysis approaches are being used. Application of these approaches to identify and phenotypically profile various cancer specific T cells in humans as well as in mouse models will be described. In addition to the possibility of finding novel therapeutic targets, a long-term goal of this work is to discover more accurate biomarkers of clinical outcomes (e.g., responsiveness to immunotherapy) based on the high-dimensional characteristics of antigen-specific T cells.

Citation Format: Evan W. Newell. Identifying and profiling tumor specific T cells using mass cytometry and highly multiplexed peptide-MHC tetramer staining. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA25.
Abstract IA35: Discovery of porcupine inhibitors targeting Wnt signaling in cancer
Authors: Pan Shifeng , Liu Jun , Cheng Dai , Han Dong , Zhang Guobao , Hsieh Mindy , Ng Nicholas , Li Chun , Kasibhatla Shailaja , McNamara Peter , Seidel H. Martin , Harris Jennifer
Published: May 2016

Wnt signaling is tightly controlled during cellular proliferation, differentiation and embryonic morphogenesis. Aberrant activation of this pathway plays a critical role in a variety of cancers. Blockade of Wnt signaling is therefore an attractive therapeutic approach for anticancer therapy. In this presentation, we will discuss our approach to search for inhibitors of Wnt ligand secretion. We developed and performed a cellular high-throughput screen using a co-culture system. Lead structure (GNF-1331) was identified and further target elucidation revealed Porcupine, a membrane bound O-acyl transferase, as its molecular target. Further structure-activity relationship studies led to the discovery of WNT974, a potent and specific Porcupine inhibitor. Treatment of WNT974 leads to tumor regression in a Wnt dependent MMTV-Wnt1 mouse model at well tolerated doses. WNT974 is currently in Phase 1 clinical trials.

Citation Format: Shifeng Pan, Jun Liu, Dai Cheng, Dong Han, Guobao Zhang, Mindy Hsieh, Nicholas Ng, Chun Li, Shailaja Kasibhatla, Peter McNamara, H. Martin Seidel, Jennifer Harris. Discovery of porcupine inhibitors targeting Wnt signaling in cancer. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA35.