bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-B20: Characterization and Enumeration of Multiple Circulating Tumor Cell Phenotypes Using Two Distinct Platforms Establishes Presence of Epithelial-Mesenchymal Transition CTCs in Patients
Authors: Wang Lihua , Owusu Francis , Khin Sonny , Parchment Ralph , Chen Alice , Kummar Shivaani , Doroshow James H. , Kinders Robert J.
Published: May 2016

We are employing a “liquid biopsy” approach for monitoring drug responses to new anti-cancer agents in early stage clinical trials by measuring drug effects in circulating tumor cells (CTCs), which requires the capability to capture and characterize CTCs from patients with diverse diseases including sarcomas, lymphomas and carcinomas. We have been applying the Apostream platform (DEP-FFF separation) with fluorescence imaging and have recently added the 5-channel CellSearch system (MAb coated ferrofluid) for specimen analysis. Use of both CD146 and EpCam capture per specimen, with addition of vimentin and the tumor markers, significantly increased the number of CTCs captured with the 5 channel CellSearch®. A “homebrew” CTC kit was developed that captures circulating cells that are either EpCAM or CD146 marker positive, and it was capable of identifying high numbers of EMT+ tumor marker+ double positive cells in patient specimens. There was statistical equivalency of events flagged as CTCs (CD45-/EpCam+/CK+) in patient specimens analyzed using Apostream or 5-Color CellSearch versus 4-channel CellSearch and 100% concordance between the CellSearch platforms. Reproducibility, as assessed by image re-analysis using Definiens software, was also high (>90%). Tumor marker specificity was established by analyzing normal donor blood, which varied from zero to 2 cells per ml blood depending on the marker set employed (only CD45 negative cells assessed). This approach solves a major limitation of using CTCs to monitor pharmacodynamic response by enumerating statistically significant cell numbers from patients with solid tumors of diverse histologies.

Strikingly, both platforms demonstrated the presence of high numbers of CTCs with the Epithelial-Mesenchymal Transition (EMT) phenotype (10-100 cells per ml in Phase 1 trial populations), and widely used tumor markers such as MUC1, CEA, TLE1, or proteins generated by recombination events such as ASPL-TFE3. The presence of large numbers of CTCs expressing the EMT phenotype may be related to the intensive prior treatment (>3 prior therapies, median) of our phase 1 population.

Funded by NCI contract # HHSN261200800001E.

Citation Format: Lihua Wang, Francis Owusu, Sonny Khin, Ralph Parchment, Alice Chen, Shivaani Kummar, James H. Doroshow, Robert J. Kinders. Characterization and Enumeration of Multiple Circulating Tumor Cell Phenotypes Using Two Distinct Platforms Establishes Presence of Epithelial-Mesenchymal Transition CTCs in Patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B20.
Abstract LB-C04: Modeling the impact of somatic alterations across human cancers
Authors: Osmanbeyoglu Hatice U. , Leslie Christina
Published: May 2016

Modeling the impact of somatic alterations across human cancers

Hatice U. Osmanbeyoglu1, Christina S. Leslie1,*

1Computational Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY

Abstract

Large-scale cancer genomics projects like The Cancer Genome Atlas have generated a comprehensive catalog of somatic mutations and copy number aberrations across many tumor types, but the role of some frequently altered genes remains obscure. To better model the impact of these alterations, we developed a computational strategy for exploiting parallel phosphoproteomics and mRNA sequencing data for large tumor sets to link dysregulation of upstream signaling pathways with altered transcriptional response through the transcriptional circuitry. Our modeling allows us to interpret the impact of mutations and copy number events in terms of altered signaling and transcription factor (TF) activity. We used a novel machine learning strategy to train phosphoprotein-TF interaction models across 10 human cancers for which large reverse-phase protein array and RNA-seq data sets are available through TCGA. We then applied a novel algorithmic approach to extract networks of signaling proteins and TFs whose inferred activities are correlated across tumors and whose dysregulation is associated with specific somatically altered genes. Our analysis revealed both known and novel interactions of frequently altered genes with signaling pathways and transcriptional programs in a pan-cancer context. Moreover, our algorithmic approach provides a general strategy for modeling the impact of recurrent mutations and copy number alterations on signaling pathways and transcriptional programs through pan-cancer analysis.

Citation Format: Hatice U. Osmanbeyoglu, Christina Leslie. Modeling the impact of somatic alterations across human cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C04.
Abstract LB-C08: Exploring glycan markers for immunotyping and precision-targeting of breast circulating tumor cells (CTCs)
Authors: Sambucetti Lidia C. , Liu Xiaohe , Hsieh Ben , Bruce Richard , Somlo George , Huang Jiaoti , Wang Denong
Published: May 2016

Recognition of abnormal glycosylation in virtually every cancer type has raised great interest in exploration of the tumor glycome for biomarker discovery. Identifying glycan markers of circulating tumor cells (CTCs) represents a new development in tumor biomarker discovery. The aim of this study was to establish an experimental approach to enable rapid screening of CTCs for glycan marker identification and characterization. We applied carbohydrate microarrays and a high-speed fiber-optic array scanning technology (FASTcell) to explore potential glycan markers of breast CTCs (bCTCs) and targeting antibodies. An anti-tumor monoclonal antibody, HAE3-C1 (C1), was identified as a key immunological probe in this study. In our carbohydrate microarray analysis, C1 was found to be highly specific for an O-glycan cryptic epitope, gpC1. Using FASTcell technology, we established a procedure to quantify expression levels of gpC1 in tumor cells. In blood samples from five Stage IV metastatic breast cancer patients, the gpC1 positive CTCs were detected in all subjects; approximately 40% of bCTCs were strongly gpC1 positive. Interestingly, the CTCs from a triple-negative breast cancer patient with multiple sites of metastasis were predominantly gpC1 positive (92.5%, 37/40 CTCs). Taken together, we present here a practical approach to examine rare cell expression of glycan markers. Using this approach, we identified an O-core glyco-determinant gpC1 as a potential immunological target of bCTCs. Given its bCTC-expression profile, this target warrants an extended investigation in a larger cohort of breast cancer patients.

Citation Format: Lidia C. Sambucetti, Xiaohe Liu, Ben Hsieh, Richard Bruce, George Somlo, Jiaoti Huang, Denong Wang. Exploring glycan markers for immunotyping and precision-targeting of breast circulating tumor cells (CTCs). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C08.
Abstract LB-C11: HTLV-1 Tax-induced apoptosis to counteract tumor drug resistance
Authors: Ducas Vanessa C. , Kupfer Gary M.
Published: May 2016

p53 mutations have been identified in over 50% of human cancers. As p53-mutant tumor cells have an inherent resistance to therapy-induced apoptosis, such a mechanism of resistance is an important problem in cancer therapy. Interestingly, Tax, a protein expressed by the human T-cell lymphotropic virus type 1 (HTLV-1), increases the sensitivity of p53-mutant and p53-null cells to DNA-damaging drugs. Tax-induced chemosensitivity presents therefore a novel strategy that can be exploited to counteract p53-mutant tumor resistance. The objective of this work is to harness the chemosensitizing property of Tax and use it to develop a chemotherapeutic approach that can overcome the drug resistance observed in p53-mutant/p53-null cancers. We hypothesize that Tax initiates a proapoptotic response in cancer cells by modulating cyclin-dependent kinase 4 (CDK4) activity. We use a combination of in vitro and in silico analyses to determine the mechanism of Tax-induced chemosensitivity and to identify small molecules that can phenocopy the CDK4-activating and the proapoptotic effects of Tax. Immunoblot analyses and in vitro kinase assays reveal that Tax alters CDK4 expression and activity. In addition, flow cytometry and gene expression studies respectively indicate that Tax expression promotes G1-to-S phase transition and leads to increased expression of the TP73 gene, a known homolog of TP53. Moreover, our in silico analyses, using an N-terminal construct of Tax and a published structure of CDK4 (PDB 3G33), reveal that CDK4 interacts with the Tax construct via a C-terminal domain that is distinct from its nucleotide-binding and D-type cyclin-binding sites. Further analyses identify ten small molecule mimics of Tax and putative CDK4 activators, which we show have a proliferative effect on treated cells. Although the focus of ongoing studies in the field has been the development of kinase inhibitors, several tumors develop strategies allowing them to override the effect of those inhibitors. Our work adopts a different strategy that will maximize the cytotoxic effect of anticancer drugs to overcome acquired drug resistance.

Citation Format: Vanessa C. Ducas, Gary M. Kupfer. HTLV-1 Tax-induced apoptosis to counteract tumor drug resistance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C11.
Abstract LB-C19: The investigation of structural determinants that mediate selective inhibition of UBA5, the UFM1 activating enzyme
Authors: Paiva Stacey-Lynn , da Silva Sara , Bancerz Mathew , Geletu Mulu , Lewis Andrew M. , Chen Jijun , Cai Yafei , Lukkarila Julie L. , Li Honglin , Gunning Patrick T.
Published: May 2016

Protein conjugation with the UFM1 small protein modifier promotes cellular survival in times of ER stress, which is a common occurrence within diseased cells. We have developed the first selective inhibitor of UBA5, the E1 activating enzyme responsible for initiating UFM1 conjugation. Based on the crystal structure of human UBA5, we identified acidic residues in close proximity to the ATP pocket that we chose to target using a zinc-coordinated polyazamacrocycle. This coordination complex was appended to adenosine through alkyl linkers of varying length in order to probe for optimal inhibition of UBA5 activity. Through this small structure-activity relationship (SAR), we identified our lead inhibitor 5C-Z that disrupts UBA5-mediated UFM1 conjugation in vitro through a non-competitive mode of action (IC50 = 4.1 μM, 95% Confidence Interval (C.I.) = 2.1-7.7 μM). Our compound demonstrates selectivity for UBA5 over other E1 enzymes in vitro, as well as over nearly 100 human kinases as evaluated using a kinome screen, despite containing adenosine within its structure. Currently, we are investigating the structural determinants that are indispensible for inhibitory activity. We are making iterative changes by varying each component of 5C-Z including the metal centre, the polyazamacrocycle, the linker, the ribose as well as the nitrogenous base. To date, we have shown that other transition metals are not as active as the zinc (II) centre, increasing the size of the macrocycle abolishes activity and reducing the linkage from the polyazamacrocycle to the linker also significantly abrogates activity. Investigating the role of each functional group on 5C-Z will help delineate the mechanism by which it selectively inhibits UBA5 activity and may shed light in progressing towards a more drug-like small molecule inhibitor.

Citation Format: Stacey-Lynn Paiva, Sara da Silva, Mathew Bancerz, Mulu Geletu, Andrew M. Lewis, Jijun Chen, Yafei Cai, Julie L. Lukkarila, Honglin Li, Patrick T. Gunning. The investigation of structural determinants that mediate selective inhibition of UBA5, the UFM1 activating enzyme. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C19.
Abstract PL07-03: The multifaceted role of Notch signaling in tumor/microenvironment interaction
Authors: Aster Jon C.
Published: May 2016

Notch receptors participate in a highly conserved signaling pathway with critical developmental roles in multicellular animals. Based on studies involving developing and adult organisms, it is evident that the outcome of Notch signaling is not fixed but varies widely with dose and context, allowing Notch to either drive or modulate a large number of different biologic processes. This complexity extends to cancer. The first mammalian Notch receptor to be identified, Notch1, was found due to its rearrangement in a rare subset of T cell acute lymphoblastic leukemia (T-ALL), immediately indicating a role for the pathway in oncogenesis. Since this time, mechanistic work in animal models and cancer genome sequencing studies have greatly expanded the scope of the proposed roles for Notch signaling in cancer to the point where it is now reasonable to think that Notch impacts all of the Weinberg/Hanahan “Hallmarks of Cancer”. Some of these roles stem from cell autonomous mutations that constitutively activate oncogenic Notch signaling in tumors such as T-ALL and triple negative breast cancer, or that inactivate Notch in tumors such as squamous cell carcinomas of the skin and head and neck, confirming that Notch can act as a context dependent oncogene or tumor suppressor. In addition, recent data suggest that certain classes of Notch mutations are selected for their ability to upregulate signaling in a microenvironment-dependent fashion, a type of gene-environment interaction that is highly specific for particular cancers, such as chronic lymphocytic leukemia and marginal zone B cell lymphoma. Additional roles for Notch involving the stromal compartment in cancers, particularly angiogenesis and host immunity, also are coming into focus. This talk will touch on a number of these emerging relationships, which have implications for ongoing attempts to effectively target the Notch pathway in patients with a broad spectrum of different cancers.

Citation Format: Jon C. Aster. The multifaceted role of Notch signaling in tumor/microenvironment interaction. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL07-03.
Abstract PR09: TAS3681, a new type androgen receptor antagonist, suppresses ligand-independent AR activation through its AR downregulation activity
Authors: Kajiwara Daisuke , Minamiguchi Kazuhisa , Seki Masanao , Mizutani Hiroya , Aoyagi Hiroki , Okajima Shigeo , Sasaki Eiji , Utsugi Teruhiro , Iwasawa Yoshikazu
Published: May 2016

Background: Inhibitors of androgen receptor (AR) signaling axis, such as enzalutamide and abiraterone, represent important advances in the management of castration-resistant prostate cancer (CRPC). However, resistance to these drugs is still occurring. Recent reports suggest that there are several mechanisms of resistance. Ligand independent AR activation such as induction of AR splice variants or c-Myc expression is a major emerging mechanism of CRPC progression. There is an urgent need for developing a novel therapy to address these unresolved issues.

Methods: For assay of androgen-independent AR transactivation, prostate cancer cells were transiently transfected with androgen-responsive reporter gene construct. The transfected cells were treated with growth factor and cytokine in steroid-depleted media, and luciferase activity was measured. To evaluate the effect of TAS3681 on AR and c-Myc protein expression, prostate cancer cells were treated with TAS3681 in steroid-depleted media. AR and c-Myc protein levels were determined by Western blot. Real-time PCR was used to analyze the mRNA levels of c-Myc and c-Myc target gene. Chromatin immunoprecipitation was performed to determine the enrichment of AR at the element.

Results: TAS3681 dose-dependently reduced AR protein levels in prostate cancer cells. In contrast to enzalutamide, TAS3681 suppressed androgen-independent AR transactivation by growth factor and cytokine. In prostate cancer cells which express full-length AR and splice variant AR-v7, TAS3681 suppressed AR-v7 target gene expression through downregulation of AR-v7 occupancy at the enhancer. Moreover, TAS3681 reduced expression of c-Myc, critical driver of androgen-independent mechanisms of prostate cancer progression, via AR downregulation activity. In addition, real-time PCR assay showed the suppression of c-Myc and c-Myc target gene mRNA levels by TAS3681 but not by enzalutamide.

Conclusion: Our findings suggest that TAS3681, a new type AR antagonist with AR downregulation activity, has a potential to overcome ligand-independent AR activation and could be a candidate of breakthrough therapy for resistance to current AR pathway target drugs.

Citation Format: Daisuke Kajiwara, Kazuhisa Minamiguchi, Masanao Seki, Hiroya Mizutani, Hiroki Aoyagi, Shigeo Okajima, Eiji Sasaki, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS3681, a new type androgen receptor antagonist, suppresses ligand-independent AR activation through its AR downregulation activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PR09.
Abstract A33: Combinatorial targeting of tumor-associated macrophages/ microglia and radiotherapy in gliomas
Authors: Akkari Leila , Quail Daniela F. , Quick Marsha L. , Huse Jason T. , Sutton James C. , Joyce Johanna A.
Published: May 2016

Eighty percent of tumors that develop in the central nervous system are malignant gliomas, with over half being glioblastoma multiforme (GBM), the most aggressive form of this disease. Unfortunately, there have been minimal improvements in the survival of patients with GBM and these tumors remain essentially incurable. Even following treatment with standard of care therapy (surgery, radiation and temozolomide), the overall 5-year survival rate of patients diagnosed with GBM is less than 5%, highlighting the urgent need for novel and alternative therapeutic strategies to treat GBM.

In GBM patients and mouse models of the disease, the major non-cancerous cell type in the glioma microenvironment is tumor-associated macrophages/microglia (TAMMs). TAMMs can account for as much as 30% of the tumor mass in human GBM, and their accumulation is associated with poor patient prognosis. We previously used an inhibitor of colony stimulating factor 1 receptor (CSF-1R), BLZ945, to target macrophages in the RCAS-PDGF-B; nestin-TVA, Ink4a/Arf null mouse model of gliomagenesis, which faithfully recapitulates the hallmarks of human GBM. We found that CSF-1R inhibition dramatically improves survival in a long-term intervention trial and markedly regresses established high-grade lesions.

To determine the translational clinical potential for CSF-1R inhibition, we designed preclinical trials in combination with radiation, which is part of the standard treatment for GBM patients. We treated established GBMs with fractionated radiation concomitant with acute, short-term BLZ945 treatment and observed that the overall survival of animals was significantly extended compared to single treatment modalities. Moreover, incorporating long-term treatment with BLZ945 in tumors treated with fractionated radiation lead to a dramatic reduction in tumor recurrence, suggesting that reversing cancer-induced M2 macrophage programming has the potential to render gliomas more prone to response to radiotherapy. Interestingly, treatment of IR-resistant gliomas with BLZ945 de novo resulted in efficient regression of the disease, which may have important translational implications. We are currently examining the molecular mechanisms by which re-educating TAMMs increases radiation sensitivity. Together these results demonstrate critical roles for CSF1R-dependent TAMMs in promoting glioma malignancy, and highlight the clinical potential for CSF-1R inhibitors.

Citation Format: Leila Akkari, Daniela F. Quail, Marsha L. Quick, Jason T. Huse, James C. Sutton, Johanna A. Joyce. Combinatorial targeting of tumor-associated macrophages/ microglia and radiotherapy in gliomas. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A33.
Abstract A45: Semaphorin 3C mediates glioma stem cell tumorigenicity and radioresistance
Authors: Yu Jennifer , Man Jianghong , Jocelyn Shoemake , Qiulian Wu , Rich Jeremy , Bao Shideng
Published: May 2016

Purpose: Different cancer cell compartments often communicate through soluble factors to facilitate tumor growth. Glioma stem cells (GSCs) are a subset of tumor cells that resist standard therapy to contribute to disease progression. How GSCs employ a distinct secretory program to communicate with and nurture each other over the non-stem tumor cell (NSTC) population is not well defined.

Methods/Materials: GSCs were enriched from patient specimens and functionally validated. We used a candidate approach to screen for secreted factors that were preferentially used by GSCs but not NSTCs. Expression was assessed by Western blot, and confirmed by immunohistochemistry and immunofluorescence in vitro and in human and mouse GBM tissues. Ligand-receptor interactions were confirmed by co-immunoprecipitation. Rac activity was assessed by Rac-GTP pulldown. Co-culture epistasis experiments were performed using knockdown and recombinant protein approaches. Glioma stem cell marker analysis (CD133, Sox2, Olig2), cell viability (ATP-dependent assays), apoptosis (TUNEL, PI/AnnexinV, cleaved caspase 3/7), self-renewal (tumorsphere formation and limiting dilution assay) and invasion (transwell migration assay) were performed. GSCs were separated in PlexinD1 hi and lo populations by FACS. Radiation (3 Gy) was delivered by Cs-137 irradiator. GSCs were used to establish orthotopic xenograft models of glioblastoma and tumor growth and animal survival were assessed.

Results: We have found that GSCs preferentially secrete Sema3C and coordinately express PlexinA2/D1 receptors to activate Rac1/NFkB signaling in an autocrine and paracrine loop to promote their own survival. Importantly, Sema3C is not expressed in neural progenitor cells (NPCs) or NSTCs. Disruption of Sema3C induced apoptosis of GSCs, but not NPCs or NSTCs, and suppressed tumor growth in orthotopic models of glioblastoma. Introduction of activated Rac1 rescued the Sema3C knockdown phenotype in vivo. Furthermore, GSCs that expressed high levels of PlexinD1 receptors were more resistant to radiation, and Sema3C and PlexinD1 were upregulated after irradiation. Together, these data support that Sema3C signaling contributes to radiation resistance.

Conclusions: Our study supports targeting Sema3C to break this GSC-specific autocrine/paracrine loop to improve glioblastoma treatment, potentially with a high therapeutic index.

Citation Format: Jennifer Yu, Jianghong Man, Shoemake Jocelyn, Wu Qiulian, Jeremy Rich, Shideng Bao. Semaphorin 3C mediates glioma stem cell tumorigenicity and radioresistance. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A45.
Abstract B01: Molecular evolutionary process of malignant glioma
Authors: Galvao Rui P. , Bassham Susan , Nelson Thomas C. , Sydes Jason , Rosenfeld Lauren K. , Cresko William A. , Zong Hui
Published: May 2016

Malignant gliomas are devastating, incurable brain tumors, with a bleak prognosis that improved little over the past decades. Many studies have been published describing genomic and transcriptomic alterations in malignant, fully formed gliomas. However, little is known of what takes place before the malignant stage, even though transformation of a normal cell into cancer is a multi-step process and, by the malignant stage, tumor cells have become very different from their original, untransformed ancestry. To gain insight into the driving forces of glioma and develop effective treatments, one must understand the entire process of tumorigenesis, for which it will be necessary to directly characterize the cell of origin of glioma at multiple time points throughout gliomagenesis.

We recently identified adult oligodendrocyte precursor cells (OPCs) as a cell of origin for malignant gliomas using a genetic mouse model that induces mutations in p53 and NF1, two of the most frequently mutated genes in human glioblastoma [Galvao et al., 2014, PNAS]. To ensure the introduction of mutations into adult but not neonatal OPCs, we used NG2-CreER that can specifically induce loxP-mediated recombination upon the administration of tamoxifen. Time course analysis revealed several distinct stages of gliomagenesis, starting with a rapid but transient hyper-proliferation shortly after tamoxifen administration, followed by a long dormancy period and ending with malignant transformation.

To further investigate the molecular changes during the pre-transforming stages, using the same mouse model, we developed a procedure based on laser capture microdissection to collect OPCs from the adult brain at distinct stages during gliomagenesis. We then used RNA sequencing to gain mechanistic insight into the successive stages of gliomagenesis. Our results show that, compared to tumor OPCs, the gene expression profile of pre-transforming mutant OPCs remains similar to that of wild-type OPCs, indicating that most transcriptional changes occur at the final transformation step. Interestingly, careful analysis revealed expression changes in hundreds of genes shortly after p53/NF1 mutagenesis. Using bioinformatics tools such as Gene Ontology (GO) terms, Gene Set Enrichment Analysis (GSEA) and Signaling Pathway Impact Analysis (SPIA), we are currently identifying the signaling pathways most affected at each time point to gain a better understanding of the molecular evolution from mutant OPCs to malignant glioma, which could provide critical insights on how to halt or even revert that process.

Citation Format: Rui P. Galvao, Susan Bassham, Thomas C. Nelson, Jason Sydes, Lauren K. Rosenfeld, William A. Cresko, Hui Zong. Molecular evolutionary process of malignant glioma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B01.
Abstract B11: The oncogenic role of I2PP2A by inhibiting NME1 in SHH medulloblastoma
Authors: Wei Yun , Kenney Anna Maria
Published: May 2016

Medulloblastoma is a pediatric brain tumor which arises in the cerebellum and is the most common childhood malignancy. The current standard of treatment for medulloblastoma (MB) is a combination of surgery, radiation and chemotherapy, which can cause lifelong side effects to MB survivors due to damage to the developing brain. Approximately 30% of human MB show aberrant Sonic Hedgehog (SHH) signaling and feature mutated SMO/PTCH1 or amplified GLI/NMYC, components of the SHH pathway. To study SHH-driven mitogenic and oncogenic signaling pathways, we use the NeuroD2-SmoA1 mouse model to investigate how downstream effectors of SHH signaling regulate cell proliferation in MB development. The NeuroD2-SmoA1 mouse closely recapitulates human SHH MB and bears constitutively active SMO in its cerebellum. The early onset and high incidence of tumors, and the adherence to human pathology make NeuroD2-SmoA1 mouse an efficient model for MB research. Utilizing this model, I have been focusing on the nuclear oncoprotein, I2PP2A, also known as endogenous inhibitor 2 of phosphatase 2A. I2PP2A has recently been proposed to be targeted in other malignant tumors such as leukemia, prostate, and lung cancers. However, whether I2PP2A play oncogenic roles in medulloblastoma has not been established yet. I found that I2PP2A protein is markedly elevated in mouse MB cytosol compared with neighboring normal cerebellum through histology and western blot approaches. In the efforts of searching molecules interacting with I2PP2A in cytoplasm, a non-metastasis marker, called NME1, stands out as the cytosol level of this protein also shows a strong induction. NME1 is a DNase that triggers cell death when tumor cells encounter the immune attack from the microenvironment. Based on the preliminary data, I hypothesize that I2PP2A sequesters NME1 in cytosol and inhibits the activation of this DNA nicking protein, thus prevents the MB cell death. My future plans are to determine the roles of I2PP2A and NME1 in tumorigenesis, test whether inhibiting I2PP2A can induce MB cell death mediated by NME1, and finally extrapolate this research to novel therapy design targeting I2PP2A in SHH medulloblastoma.

Citation Format: Yun Wei, Anna Maria Kenney. The oncogenic role of I2PP2A by inhibiting NME1 in SHH medulloblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B11.
Abstract B20: Targeting Olig2 co-regulators for malignant glioma therapy
Authors: Kupp Robert , Cascio Costanza Lo , Rauf Femina , Meijer Dimphna , Stiles Charles D. , Sanai Nader , LaBaer Joshua , Mehta Shwetal V.
Published: May 2016

The transcription factor OLIG2 is expressed within the highly tumorigenic sub-population of stem-like cells in malignant glioma and required for tumor formation in orthotopic grafts of human glioma as well as genetically defined murine glioma models. Our work (recently confirmed and extended by Suva et al., 2014) suggests that small molecule OLIG2 antagonist could serve as targeted therapy for high grade gliomas – either as a mono therapeutic or as an adjuvant to conventional radiotherapy or cytotoxic drugs. However, transcription factors in general are considered to be unattractive targets for drug development because their interactions with DNA and co-regulatory proteins involve large and complex surface area contacts. Our recent data suggests that OLIG2 interacts with distinct partner proteins based on its phosphorylation state, which in turn regulates its pro-mitogenic functions. Here we employed two independent proteomics screens (mass spectrometry and Nucleic Acid Programmable Protein Arrays, NAPPA) to identify OLIG2 co-regulators as possible surrogate targets to suppress OLIG2 function. These screens led to identification of two novel OLIG2 partner proteins: HDAC1 and proto-oncogene FYN kinase. OLIG2 is known to be a negative regulator of gene expression and histone deacetylation is required for suppression of gene expression. We demonstrate that OLIG2 functions as an HDAC1 carrier, delivering this enzyme to specific target genes (e.g., p21) and non-histone proteins like p53. HDACs are ubiquitously expressed and are known to be redundant, but we show that ablation of HDAC1 alone (using shRNA) significantly decreases the proliferation of glioma initiating cells with minimal impact on normal neural stem cells. In addition, we show that FYN kinase interacts with OLIG2 and phosphorylates it at tyrosine residues. Ablation of FYN decreases phosphorylation of OLIG2 at tyrosine residues and affects OLIG2-mediated expression of oncogenic target genes like EGFR and WNT5a. This work provides two potential co-regulators of OLIG2 which could serve as druggable surrogate targets to block OLIG2 function in malignant glioma. Further work will evaluate the efficacy of HDAC1 and FYN-specific inhibitor as single agents or as adjuvant therapy to radiation and temozolomide in orthotopic patient-derived xenograft models. Targeting factors regulating OLIG2-mediated tumor growth will have practical overtones for targeted therapies for malignant gliomas.

Citation Format: Robert Kupp, Costanza Lo Cascio, Femina Rauf, Dimphna Meijer, Charles D. Stiles, Nader Sanai, Joshua LaBaer, Shwetal V. Mehta. Targeting Olig2 co-regulators for malignant glioma therapy. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B20.
Abstract IA02: Molecular pathogenesis of IDH mutant cancers and evolving treatment strategies
Authors: Kaelin William G.
Published: May 2016

IDH1 and IDH2 mutations have now been described in a variety of cancers including gliomas, acute myelogenous leukemias, chondrosarcomas, cholangiocarcinomas, and angioimmunoblastic T-cell lymphoma. Tumor-associated IDH1 and IDH2 mutants produce large amounts of the R enantiomer of 2-hydroxyglutarate (R-2HG), which accumulates to mM levels in IDH mutant tumor cells. R-2HG modulates (usually inhibits) various 2-oxoglutarate-dependent enzymes, including various 2-oxoglutarate-dependent dioxygenases. Examples of such dioxygenases include the JmjC family of histone demethylases and the TET methylcytosine hydroxylases. The challenge for the field has been to determine which enzymes, when modulated by R-2HG, drive transformation and which are simply bystanders. We developed a cell-based assay for hematopoietic transformation by mutant IDH and showed that transformation in this setting is indeed mediated by R-2HG and that its effects are reversible. Moreover, we and others provided evidence that inhibition of TET2 by R-2HG contributes to leukemic transformation in cells carrying IDH mutations. Small molecules that block R-2HG production appear promising based on early Phase 1 data in IDH1 and IDH2 mutant leukemias. We also developed a model of astrocytic transformation by IDH. We found that activation of the EglN prolyl hydroxylases, which target the HIF transcription factor for destruction, by R-2HG promotes soft agar growth by immortalized astrocytes but is not sufficient to promote tumor growth in immunocompromised mice. More recently we have developed an oncogene cooperation assay in which mutant IDH promotes tumor growth in mice. In addition, we have identified an enzyme in glioma cells that, when inhibited by R-2HG, creates a distinctive metabolic profile and renders IDH mutant cells dependent on glutaminase activity for survival.

Citation Format: William G. Kaelin, Jr. Molecular pathogenesis of IDH mutant cancers and evolving treatment strategies. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr IA02.
Abstract IA23: Medulloblastoma subgroup-specific mutations model the tumor microenvironment
Authors: Gilbertson Richard J.
Published: May 2016

Brain tumors, especially those arising in children, are remarkably heterogeneous in their response to treatment. Understanding the processes that dictate treatment sensitivity is important if we are to cure all patients. Most research has focused on identifying tumor cell intrinsic properties that regulate treatment response; but the role of the tumor microenvironment is less well understood. Here, we show that distinct subtypes of medulloblastoma possess markedly different vasculatures that are determined non-autonomously by tumor genotype, and that dictate response to systemic chemotherapy. We found that WNT-antagonists secreted by WNT-medulloblastoma – an essentially curable form of the disease – silence WNT signaling in neighboring endothelial cells, causing the formation of fenestrated and highly permeable vessels normally associated with peripheral organs: endothelial WNT signaling is essential to form and maintain the blood brain barrier (BBB). Consequently, systemically delivered chemotherapy accumulated to high levels in WNT-medulloblastomas, eliciting a marked clinical response. In contrast, while cells derived from Sonic Hedgehog (SHH)-medulloblastoma – a much less curable subtype – were intrinsically chemosenstive, they did not secrete WNT-antagonists, formed tumors with an intact, chemo-impermeable BBB and were chemoresistant in vivo. Remarkably, reactivation of WNT-signaling in WNT-medulloblastoma endothelial cells restored BBB function, rendering these tumors chemoresistant. Our data provide direct evidence that brain tumor genotype can dictate tumor vessel phenotype in a non-autonomous fashion, providing a potential explanation for the excellent prognosis of WNT-medulloblastoma. Pharmacological inhibition of endothelial WNT signaling in other primary and secondary brain tumors might enhance chemoresponsiveness by disrupting the BBB.

Citation Format: Richard J. Gilbertson. Medulloblastoma subgroup-specific mutations model the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr IA23.
Abstract A33: Mediator is an intrinsically dynamic complex critical to Hsf1-activated transcription
Authors: Anandhakumar Jayamani , Moustafa Yara , Chowdhary Surabhi , Kainth Amoldeep S. , Gross David S.
Published: May 2016

Mediator is an evolutionarily conserved transcriptional coactivator that plays a vital role in the regulation of RNA polymerase II (Pol II) transcribed genes in eukaryotes as diverse as yeast and human. It consists of a tri-modular core complex comprised of Head, Middle and Tail modules and a dissociable Cdk8/Kinase module (CKM). Although it has been assumed that core Mediator in the model eukaryote S. cerevisiae (budding yeast) exists as a stable 21-subunit complex based on cell-free assays, its structural state in vivo is unclear. We have obtained evidence that the Tail module is highly dynamic, and that a subcomplex consisting of Tail subunits Med2, Med3 and Med15 can be independently recruited to the UAS/promoter regions of Heat Shock Factor 1 (Hsf1)-regulated genes in response to acute thermal stress. Med2-Med3-Med15 autonomy is revealed by ChIP analysis following the “anchoring away” of select Mediator core subunits located in either Middle or Tail module. Such conditional depletion leads to a 60-80% reduction in HSP gene transcription. We additionally observed that Hsf1 can recruit the CKM independently of the core complex, and that both core Mediator and CKM recruitment occurs independently of SAGA. Collectively, our results indicate that yeast Mediator is intrinsically dynamic and that multiple species, including CKM-Mediator, the 21-subunit core complex, the Med2-Med3-Med15 Tail Triad and the four-subunit CKM, can be independently recruited by Hsf1 to its target genes.

Citation Format: Jayamani Anandhakumar, Yara Moustafa, Surabhi Chowdhary, Amoldeep S. Kainth, David S. Gross. Mediator is an intrinsically dynamic complex critical to Hsf1-activated transcription. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A33.
Abstract B10: Increased expression of EZH2 and TOP2A predicts for a poorer prognostic outcome in Genitourinary Cancers
Authors: Rosario Spencer , Dalimov Zafardjan , Kirk Jason , Morrell Kayla , Eng Kevin , Ellis Leigh
Published: May 2016

The enhancer of Zeste homolog 2 (EZH2), a component of the Polycomb Repressive Complex 2 (PRC2), is an epigenetic silencer that is known to be highly expressed in both metastatic and primary prostate cancer (PCa). More recently, topoisomerase IIα (TOP2A), an enzyme that controls the topologic states of DNA during the cell cycle, has also been identified as being highly expressed in PCa. Both EZH2 and TOP2A have been associated with more aggressive disease and poorer prognostic outcome. During genome-wide analysis, using The Cancer Genome Atlas (TCGA), EZH2 and Top2A were found to be highly positively correlated, most noticeably in PCa and clear cell Renal Cell Carcinoma (ccRCC). As a result, we predicted increased expression of both EZH2 and TOP2A in an individual, would predict for a worse prognostic outcome than either gene being highly expressed alone. Further analysis of the TCGA and Memorial Sloan Kettering Cancer Center (MSKCC) datasets, demonstrated that increased expression of these two genes predicted for a poorer prognostic outcome in genitourinary cancers. In tumor samples with increased expression of EZH2 and TOP2A, as compared to the tumor samples that did not concurrently overexpress both EZH2 and TOP2A, patients displayed worse overall Regression Free Survival (RFS), which were associated with increase hazard ratio, and tumor staging and grades. . As these poor prognostic markers were consistent across multiple cancer subtypes, we are beginning to explore those genes that are differentially expressed in individuals with concurrent high TOP2A and EZH2 as a means of further characterizing this novel phenotype. Since these two genes are targetable by drugs such as etoposide (a TOP2 poison) and DZNep (an EZH2 inhibitor), it makes them attractive candidates for treatment in those individuals who not only have tumors that highly express these two genes, but also those individuals that have exacerbated existing treatment options.

Citation Format: Spencer Rosario, Zafardjan Dalimov, Jason Kirk, Kayla Morrell, Kevin Eng, Leigh Ellis. Increased expression of EZH2 and TOP2A predicts for a poorer prognostic outcome in Genitourinary Cancers. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B10.
Abstract B14: Use of DNA methylation in circulating free DNA as a potent biomarker for pancreatic cancer detection
Authors: Shinjo Keiko , Kondo Yutaka
Published: May 2016

Pancreatic cancer is highly lethal malignancy despite the recent clinical advancement. Since pancreatic cancer is often difficult to diagnose, there is an urgent need to develop novel biomarkers for early detection with blood tests. Epigenetic alterations have emerged as a common hallmark of many types of human cancers as well as pancreatic cancer. Studies have shown that epigenetic dysregulation is associated with multiple steps during carcinogenesis. Therefore, invention of blood-based DNA methylation biomarkers and their high-sensitive detection system may be a promising diagnostic tool for pancreatic cancer. In order to establish the diagnostic system, first, we performed genome-wide DNA methylation analysis of 38 EUS-FNA specimens of pancreatic cancer with clinical stage III-IV using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the DNA methylation profiles of other types of tumors in the public database, TCGA (The Cancer Genome Atlas), and identified five marker genes, which were specifically methylated in pancreatic cancers. In addition, analysis of DNA methylation status of pancreas cancers comparing 10 normal tissues in TCGA data set revealed that 99% of tumors were methylated at least one of the five markers (sensitivity, 98.9%; specificity, 44%). We next examined the DNA methylation status in the markers in the validation cohort (n=19) by bisulfite pyrosequencing and quantitative methylation specific PCR and found that 18/19 (95%) cases were methylated in at least one marker (AUC=0.95). Currently, our preliminary data showed that more than 95% of cancer patients have aberrant DNA methylation in circulating free DNA (cfDNA). Our results indicate that detection of DNA methylation in the cfDNA might be a promising and potent tool for diagnosis of pancreatic cancers.

Citation Format: Keiko Shinjo, Yutaka Kondo. Use of DNA methylation in circulating free DNA as a potent biomarker for pancreatic cancer detection. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B14.
Abstract B26: RRx-001, a novel epigenetic modulator: Resensitization to previously failed therapy in the ongoing phase 2 colorectal cancer study, ROCKET
Authors: Reid Tony , Fisher George , Carter Corey , Cho-Phan Cheryl , Kunz Pamela , Oronsky Bryan , Fanger Gary , Stirn Meaghan , Caroen Scott , Parker Christopher , Scicinski Jan
Published: May 2016

Background: The development of resistance to chemotherapies in cancer leads to disease progression resulting in impaired survival. Resensitization to these previously effective but now failed therapies could lead to improvements in patient survival and response. RRx-001, a novel dinitroazetidine ROS-mediated epigenetic modulator that has demonstrated activity against DNMTs and HDACs may resensitize colorectal cancer patients to previously effective irinotecan therapies resulting in improvements in survival.

Methods: The ROCKET study is a two stage trial that investigates RRx-001-mediated resensitization to irinotecan therapies. Patients with metastatic colorectal cancer who previously responded, then progressed on irinotecan-based therapies, with or without bevacizumab, cetuximab or panitumumab and an ECOG PS 0-1 are randomized in Stage 1 to receive RRx-001 1xweek or regorafenib until progression or unacceptable toxicity. Patients then advance to Stage 2 to receive irinotecan-based therapies. Target accrual is approx. 190 patients and the primary endpoint is overall survival with secondary endpoints to investigate resensitization. Recruitment is ongoing.

Study Status and Results: To date, 28 patients have been randomized with 20 entering the resensitization stage of the study. 13 post RRx-001 patients entered the second stage of the study showing an improving patient survival, marked decreases in CEA and radiological responses. In contrast, the 7 regorafenib patients eligible for stage 2 were too systemically unwell to proceed to subsequent treatment.

Conclusions: Early results suggest that RRx-001, a systemically non-toxic epigenetic agent, may resensitize colorectal cancer patients to irinotecan-based therapies. These interesting early results appear to be generalized and may translate to increased overall survival, adding an additional regimen after disease progression. The trial is continuing.

Citation Format: Tony Reid, George Fisher, Corey Carter, Cheryl Cho-Phan, Pamela Kunz, Bryan Oronsky, Gary Fanger, Meaghan Stirn, Scott Caroen, Christopher Parker, Jan Scicinski. RRx-001, a novel epigenetic modulator: Resensitization to previously failed therapy in the ongoing phase 2 colorectal cancer study, ROCKET. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B26.