bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2658: Genome-wide mistargeting of oncogenic SWI/SNF(BAF) complexes in SMARCB1(BAF47)-deficient sarcomas
Authors: Nakayama Robert , Williams Robert T. , Cassel Seth H. , Teng Mingxiang , Irizarry Rafael A. , Demetri George D. , Kadoch Cigall
Published: July 2016

SMARCB1/BAF47/INI1 is a core subunit of the mammalian SWI/SNF (BAF) family of ATP-dependent chromatin remodeling complexes, which remodel nucleosome architecture to achieve coordinated regulation of gene expression. Genetic loss of SMARCB1 has been identified in several cancer types, including malignant rhabdoid tumor (MRT, 98%) and epithelioid sarcoma (EpS, 90%), strongly implicating this event as the oncogenic driver in these malignancies. However, the precise mechanism underpinning the tumor suppressive function of BAF47 to date remains unclear. In order to elucidate the underlying mechanism and to identify direct genetic targets of aberrant BAF complexes in this context, we comprehensively evaluated the effects of BAF47 reintroduction in BAF47-deficient sarcomas with respect to complex subunit and associated protein factor composition and stability, global chromatin structure, and gene regulation.

Reintroduced BAF47 stably integrated into BAF complexes, and remarkably, stabilized a highly specific set of BAF subunits, resulting in an increased complex molecular weight and stoichiometric nuclear abundance. These biochemical changes inducing the formation of wild-type complexes in MRT and EpS cell settings were directly linked to reproducible changes in BAF complex localization genome-wide, particularly, in the targeting to H3K4me3-marked promoter regions of direct target genes to establish DNA accessibility. Importantly, changes in BAF47-dependent BAF complex targeting between oncogenic and induced wild-type conditions were reproducibly associated with differential chromatin architecture and gene expression signatures hallmark to both MRT and EpS, and uniformly resulted in proliferative senescence of MRT and EpS cell lines in culture.

These studies highlight, for the first time, the full spectrum of structural and functional contributions of the BAF47 subunit, implicating its role as a keystone in heteromorphic BAF complex assembly; BAF47 is required for the stable integration of several BAF subunits and novel interacting factors, which we determine govern specific genome-wide targeting mechanisms and chromatin-templated activities. These results reveal the mechanisms underlying the oncogenesis of BAF47-deficient sarcomas and point toward novel therapeutic strategies for this group of human sarcomas.

Citation Format: Robert Nakayama, Robert T. Williams, Seth H. Cassel, Mingxiang Teng, Rafael A. Irizarry, George D. Demetri, Cigall Kadoch. Genome-wide mistargeting of oncogenic SWI/SNF(BAF) complexes in SMARCB1(BAF47)-deficient sarcomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2658.
Abstract 2663: Insights into the oncogenic basis of long non-coding RNA PVT1 in human cancer
Authors: Tashiro Kojiro , Klink Robert , Zetterval Jon , Diedrich Veronica , Tseng Yuen-Yi , Bagchi Anindya
Published: July 2016

Though gain of 8q24.21 is a common mutation in human cancers, its functional annotation is limited to studying myelocytomatosis (MYC), the prominent oncogene in the amplicon. However, MYC is co-gained with an adjacent long non-coding RNA gene plasmacytoma variant translocation 1 (PVT1), the CCDC26 gene candidate and gasdermin C GSDMC. Whether copy number gain of one or more of these genes drives neoplasia is not known. We developed chromosome engineered mouse strains with an extra copy of 1) Myc, 2) Pvt1, Ccdc26, Gsdmc, and 3) Myc, Pvt1, Ccdc26, Gsdmc. When rat Neu was introduced to test the change in the latency in mammary tumor development, only the mice with an extra copy of Myc, Pvt1, Ccdc26, Gsdmc (but not with an extra copy of Myc or Pvt1, Ccdc26, Gsdmc) developed adenocarcinomas with reduced latency, suggesting that while an extra copy of Myc gene failed to measurably advance cancer, it may co-operate with Pvt1, Ccdc26 or Gsdmc to promote cancer. Si-RNA mediated knockdown of Pvt1/PVT1 reduced the proliferation rates in the tumors, and in SK-BR-3 and MDA-MB-231, two human breast cancer cell lines with 8q24 amplification. Ablation of PVT1 markedly decreased MYC protein levels, suggesting a PVT1-dependence of MYC protein in MYC amplified cancer cells. Analysis of the cancer genome atlas suggested 99% of tumors with increased MYC copy-number also harbor co-gain of PVT1. Tissue microarray analysis revealed that PVT1 RNA and MYC protein expression are correlated in primary human tumors. These data suggests that PVT1 can potentiate MYC in human cancers. CRISPR mediated deletion of PVT1 in colorectal cell line HCT116 impaired tumor formation in xenografts, and significantly reduced MYC levels. We further confirmed the oncogenic potential of PVT1 in cancers even without supernumerary 8q24. Additionally, we have identified the putative functional domain of PVT1 which confers its oncogenic potential. We propose that the dependence of high levels of MYC on lncRNA PVT1 provides a much-needed therapeutic window against MYC protein, known to be refractory to small molecule inhibition.

Citation Format: Kojiro Tashiro, Robert Klink, Jon Zetterval, Veronica Diedrich, Yuen-Yi Tseng, Anindya Bagchi. Insights into the oncogenic basis of long non-coding RNA PVT1 in human cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2663.
Abstract 2669: Glutaminase alternative splicing controls tumor growth and metabolism
Authors: Stine Zachary , Walton Zandra E. , Zhang Lin , Fan Teresa MW , Lane Andrew N. , Dang Chi V.
Published: July 2016

Cancer cells reprogram their metabolism to meet their energetic and biosynthetic needs, with many cancer cells showing increased aerobic glycolysis and glutamine consumption. In addition to providing carbon to the TCA cycle to compensate for the diversion of glucose to lactate, glutamine is an important carbon and nitrogen donor for the synthesis of nucleotides, amino sugars and non-essential amino acids. Glutaminase (GLS) catalyzes the first step of glutamine metabolism, the hydrolysis of glutamine to glutamate, which can then be converted to alpha-ketoglutarate, glutathione or non-essential amino acids. GLS is alternatively spliced to make the KGA or more catalytically active GAC isoforms. Analysis of publicly available TCGA RNA-seq data from primary human tumors shows that GAC is the predominant splice form of GLS in many tumor types. However, the roles played by GAC versus KGA in tumor metabolism and progression are poorly understood. To facilitate the study of GLS alternative splicing, CRISPR/Cas9 mediated deletion of the GAC specific exon was used to convert predominantly GAC-expressing parental cell lines to solely KGA-expressing cell lines (ΔGAC). Despite showing decreased glutamine-derived glutamate and α-ketoglutarate, ΔGAC cells do not show impaired growth in nutrient-replete in vitro conditions compared to parental cells. 13C-isotopic labeling and metabolomics showed that ΔGAC cells have decreased levels of proline and alanine, both of which can be derived from glutamine-derived glutamate. This led us to ask if ΔGAC cells are dependent on exogenous non-essential amino acids. We found that depletion of non-essential amino acids slowed the growth of ΔGAC cells compared with parental cells, rescuable by exogenous proline and alanine. Importantly, the growth of lung (A549) and breast cancer (MDA-MB231) ΔGAC xenograft models was diminished compared with parental tumor xenografts in vivo, which is consistent with a role for GAC in nutrient challenged conditions. In summary, the GAC form of glutaminase fuels tumor growth partly through synthesis of non-essential amino acids, particularly under nutrient limiting conditions. As GLS inhibitors enter the clinic, a more thorough understanding of the role of GLS in tumor metabolism will aid in the development of treatment strategies to best make use of these drugs.

Citation Format: Zachary Stine, Zandra E. Walton, Lin Zhang, Teresa MW Fan, Andrew N. Lane, Chi V. Dang. Glutaminase alternative splicing controls tumor growth and metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2669.
Abstract 2681: Intravital microscopy of prostate cancer lesions in bone: kinetics of osteolysis and therapy response
Authors: Dondossola Eleonora , Alexander Stephanie , Holzapfel Boris , Logothetis Christopher J. , Hutmacher DIetmar , Friedl Peter
Published: July 2016

Bone metastases are the initial site of progression and account for many of the complications experienced by men with metastatic prostate cancer (PCa), including therapy resistance. Besides cell intrinsic mechanisms, the interaction between PCa cells and the bone microenvironment critically contributes to lesion expansion and drug sensitivity. We here developed a mouse model amenable to intravital multiphoton microscopy (iMPM) to longitudinally study PCa-stromal cell interactions and therapy response in a partially humanized neobone, established in the dermis of the mouse. Tissue engineered bone constructs, TEBCs, were generated by functionalizing polymeric polycaprolactone scaffolds with human mesenchymal stem cells (hMSCs) differentiated to osteoblasts followed by cell-derived calcification of the inter-scaffold space. 30 days after implantation of functionalized scaffolds under the skin of the mouse, bone and bone marrow maturation resulted in a 30mm3 cavity surrounded by optically transparent cortical bone of 50-60 μm thickness, as monitored by microCT and iMPM. Intra-bone tumor growth and osteolysis caused by human fluorescent PCa cells (PC3) were three-dimensionally reconstructed by multi-parameter detection through a body window, including collagen and bone matrix (SHG), calcified bone (fluorescent bisphosphonates), osteoclasts (cathepsin K), bone surface (THG), blood vessels and stromal phagocytes (fluorescent dextran), and PC3 cells (nuclear H2B/eGFP, cytoplasmatic DsRed2). As a proof of concept, the efficacy to halt bone remodeling during bisphosphonate therapy was detected, revealing the composition and kinetics of the bone-tumor interface, including oscillating bone loss followed by neo-apposition. Thus, combining engineered neobone with an optical window is suited to detail the 3D organization and dynamics of cancer lesions in bone and provides mechanistic insight and efficacy predictions for therapy response.

Citation Format: Eleonora Dondossola, Stephanie Alexander, Boris Holzapfel, Christopher J. Logothetis, DIetmar Hutmacher, Peter Friedl. Intravital microscopy of prostate cancer lesions in bone: kinetics of osteolysis and therapy response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2681.
Abstract 2695: Targeted therapy of leukemia and Ewing's sarcoma by human antibody-targeted nanoparticles
Authors: Kang Hyunggyoo , Nagy Jon , Trivhe Timothy
Published: July 2016

Current cancer therapies have shown limited success due to the poor selectivity. The recent chemotherapy could be far more effective if higher doses could be specifically delivered to the tumor and not to normal tissues. Therefore a reliable tumor specific therapy is urgently needed to treat cancer patients. Targeted nanoparticle can attenuate the off-target toxicity and enhance the efficacy of existing therapies, which would be highly beneficial to the cancer patients. In spite of a lot of effort still the optimal formulation of nanoparticle for the actual translational applications was not established yet. Liposomes, unilamellar vesicles composed of natural and/or synthetic lipids have been an intensively studied system. By refining the early formulations and after extensive testing, recently we could invent a new type of lipid bilayer nanoparticle delivery system, HPLN (Hybrid Polymerized Liposomal Nanoparticles) and demonstrate that this new type of polymeric nanoparticle overcomes many, if not most, of the deficiencies noted and has tremendous potential as an effective delivery vehicle for treating many types of cancer. In our study it was shown that the targeted HPLN with human antibodies (antiCD99 or antiCD19) can inhibit the growth of Ewing's sarcoma and leukemia (ALL) in a murine model. The antitumor effects of of HPLN were diminished pretty much by the removal of antibody or drug (Doxorubicin), which proves efficacy of the targeted HPLNs is specifically dependent upon the targeting antibody and drug. In addition, no abnormalities in liver and kidney function tests, complete blood counts or pathology of major organs are observed from tail-vein administrations. These data provide strong evidence for the safety and efficacy of this targeted HPLN delivery system of anticancer drugs to Ewing's sarcoma and leukemia (ALL). In conclusion, our result showed a potential of targeted HPLN as a selective delivery vehicle of cytotoxic drugs to cancers. Hopefully this technology will be applied to treat many other different types of tumor cells in the future.

Citation Format: Hyunggyoo Kang, Jon Nagy, Timothy Trivhe. Targeted therapy of leukemia and Ewing's sarcoma by human antibody-targeted nanoparticles. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2695.
Abstract 2699: A cancer treatment based on synergy between anti-angiogenic and immune cell therapies
Authors: Soto-Ortiz Luis F.
Published: July 2016

A mathematical model integrating tumor angiogenesis and tumor-targeted cytotoxicity by immune cells was developed to identify the therapeutic window of two distinct modes to treat cancer: 1) an anti-angiogenesis treatment based on the monoclonal antibody bevacizumab that targets tumor vasculature, and 2) immunotherapy involving the injection of unlicensed dendritic cells to boost the anti-tumor adaptive response. The angiogenic cytokine Vascular Endothelial Growth Factor (VEGF) contributes to the immunosuppressive tumor microenvironment, which is responsible for the short-lived therapeutic effect of cancer-targeted immunotherapy. The effect of immunosuppression on the width of the therapeutic window of each treatment was quantified. Experimental evidence has shown that neutralizing immunosuppressive cytokines results in an enhanced immune response against infections and chronic diseases. The model was used to determine treatment protocols involving the combination of anti-VEGF and unlicensed dendritic cell injections that enhance tumor regression. The model simulations predicted that the most effective method to treat tumors involves administering a series of biweekly anti-VEGF injections to disrupt angiogenic processes and limit tumor growth. The simulations also verified the hypothesis that reducing the concentration of the immunosuppressive factor VEGF prior to an injection of unlicensed dendritic cells enhances the cytotoxicity of CD8+ T cells and results in complete tumor elimination. Feasible treatment protocols for tumors that are diagnosed late and have grown to a relatively large size were identified.

Citation Format: Luis F. Soto-Ortiz. A cancer treatment based on synergy between anti-angiogenic and immune cell therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2699.
Abstract 2700: Transcriptome analyses reveal fusion transcripts and transcript variants that are recurrent across sample series of testicular germ cell tumors
Authors: Hoff Andreas M. , Zhao Sen , Johannessen Bjarne , Skotheim Rolf I.
Published: July 2016

Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men. Embryonal carcinoma (EC) comprises a histological subgroup of TGCT that exhibits pluripotent characteristics similar to embryonic stem (ES) cells. The genetic drivers underlying malignant transformation of TGCT, including ECs, are unknown. To elucidate the abnormal genetic events potentially contributing to TGCT malignancy, such as the formation of fusion genes or expression of aberrant transcript variants, we have previously performed RNA sequencing of EC cell lines and their non-malignant ES cell line counterparts. From this dataset, we identified and characterized eight novel fusion transcripts and one gene with alternative promoter usage, ETV6. These were the first fusion genes to be shown in TGCT (Hoff et al., Cancer Research, in press).

To search for recurrence of the originally nominated TGCT specific fusion transcripts and other transcript variants in an external sample series, we have here parsed raw RNA sequencing data from 102 primary TGCTs obtained from The Cancer Genome Atlas (TCGA; unpublished). In this process, we have evaluated multiple fusion finder algorithms and approaches to determine the optimal method, on the basis of sensitivity and specificity, to query specific transcript variants and breakpoints in RNA sequencing datasets.

We were able to detect the recurrent fusion transcripts; RCC1-ABHD12B, RCC1-HENMT1, CLEC6A-CLEC4D and the transcript variant of ETV6, which was previously detected from our in-house RNA sequencing dataset from EC cell lines. We also demonstrate that expression of several fusion transcripts correlate significantly with the histological subtype. In conclusion, the novel fusion transcripts and ETV6 transcript variant are expressed from a high fraction of primary TGCTs with low differentiation grade, as detected across independent sample series.

Citation Format: Andreas M. Hoff, Sen Zhao, Bjarne Johannessen, Rolf I. Skotheim. Transcriptome analyses reveal fusion transcripts and transcript variants that are recurrent across sample series of testicular germ cell tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2700.
Abstract 2705: A computational algorithm to predict tumor growth and cancer stem cell proportion in-vitro and in-vivo from single-cell observations
Authors: Pearson Alexander T. , Ingram Patrick , Bai Shoumei , Yoon Euisik , Jackson Trachette , Buckanovich Ronald J.
Published: July 2016

We have developed a new mathematical algorithm and corresponding computer software that uses observations of single cell behavior to predict the growth of the cancer stem-like cell proportion in larger cancer cell groups. Cancer stem-like cells (CSCs) have been implicated in ovarian cancer tumor growth, chemotherapy resistance, and disease recurrence. Aldehyde dehydrogenase (ALDH) is a primary discriminator of CSCs in ovarian cancer as well as in other cancer types. Unfortunately, the rarity and ability of CSCs to rapidly differentiate makes them difficult to study in-vitro and in-vivo. Microfluidic capture devices now allow us to grow and evaluate single ovarian cancer cells in isolated culture. We deployed microfluidic devices to evaluate the different self-renewal and asymmetric division patterns of ALDH+ and ALDH(-) ovarian CSCs. We analyzed data gathered from arrays of single cell chamber observations and found that purified ALDH+ cells were more proliferative than ALDH(-) cells in both cell-line (n = 112, p < 0.001) and primary (n = 41, p = 0.008) ovarian cancer specimens. Importantly, ALDH+ cells could produce both ALDH+ and ALDH(-) cells, whereas ALDH- cells were only able to produce ALDH- cells. Based on this hierarchy-defining data, we developed an easy to use computer algorithm on the freely-available software R to predict cancer cell population growth in-vitro and in-vivo. In our algorithm, size changes of cell populations are simulated by drawing from observed events. We compared the predictions from our hybrid microfluidics chip and computational algorithm with validation experiments in-vivo and in-vitro for both cell line and primary ovarian cancer cells. We found a superb correlation between observed and predicted in-vitro total and CSC counts for both cell line and primary ovarian cancer cells (correlation r = 0.98, p < 0.0001). Furthermore, this approach appropriately predicted changes in cell growth in the presence of the CSC-promoting growth factor EGFL6 both in vitro and in vivo, over time frames of up to 28 days. The single cell division based sampling algorithm developed here allows for rapid and inexpensive means to predict in-vivo ovarian tumor growth based on microfluidic chip culture and can easily be adapted to evaluate new therapeutic options across other cancer subtypes.

Citation Format: Alexander T. Pearson, Patrick Ingram, Shoumei Bai, Euisik Yoon, Trachette Jackson, Ronald J. Buckanovich. A computational algorithm to predict tumor growth and cancer stem cell proportion in-vitro and in-vivo from single-cell observations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2705.
Abstract 2707: Quantitative analysis of the role of androgen receptor in radiation treatment for prostate cancer
Authors: Qian Mengdi , Almasan Alexandru , Gurkan-Cavusoglu Evren
Published: July 2016

The purpose of this study is to quantitatively investigate the mechanism of androgen receptor (AR) and non-homologous end joining (NHEJ) interaction after and ionizing radiation (IR) and androgen deprivation therapy (ADT) treatment. In most clinical treatment approaches, a combination of ADT and IR is used in order to improve prostate patients’ overall survival probability. It is found that AR interacts with NHEJ pathway, a major double strand break (DSB) repair pathway. We have developed a mathematical model of the NHEJ pathway that is composed of a series of ordinary differential equations. The kinetic rate constants constitute the unknown parameters of the model and we have estimated these parameters using least square estimation. The data sets used in the parameter estimation were obtained from the literature and they were from both in vitro experiments as well as clinical data from prostate cancer patients. Both sets of data demonstrated the effects of AR on DSB repair by NHEJ. The in vitro data suggest that AR enhances DSB repair by inducing expression/activity of DNA-PKcs, which is a key protein in NHEJ. The clinical data show that the effect is through the first NHEJ protein complex that is recruited to DSB, Ku70/80. We have developed the models for IR treatment alone or in combination with ADT to determine which of these mechanisms can explain the behavior in both data sets simultaneously. We have carried out sensitivity analysis and identifiability tests on all the parameters from both models in order to determine the parameters that are reliably estimated. We have shown that when the rate constant for the initiation protein Ku70/80 is decreased to half in the IR+ADT case compared to the IR only case, the model outputs captured the observations in both experimental and clinical data. The rate constant for Ku70/80 is proved to be both sensitive and identifiable, indicating that the two-fold difference in the rate constants of IR only and IR+ADT cases is biologically relevant. Therefore, we conclude that when ADT is applied, AR significantly slows down the kinetic activity of Ku70/80, thus the DSB repair by NHEJ. The suppression of AR activity through ADT has the potential to enhance the IR treatment outcomes for the prostate cancer patients that are resistant to IR-only treatment.

Citation Format: Mengdi Qian, Alexandru Almasan, Evren Gurkan-Cavusoglu. Quantitative analysis of the role of androgen receptor in radiation treatment for prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2707.
Abstract 2725: Probing mitotic functions of BUB1 kinase using the small molecule inhibitors BAY-320 and BAY-524
Authors: Baron Anna P. , von Schubert Conrad , Cubizolles Fabien , Siemeister Gerhard , Hitchcock Marion , Mengel Anne , Schröder Jens , Fernández-Montalván Amaury , Lange Martin , von Nussbaum Franz , Mumberg Dominik , Nigg Erich
Published: July 2016

The maintenance of correct chromosome number (euploidy) during cell division is ensured by a highly conserved surveillance mechanism termed ‘spindle assembly checkpoint’ which safeguards correct chromosome segregation by delaying anaphase onset until all chromosomes are properly bi-oriented on the spindle apparatus. The mitotic kinase BUB1 (budding uninhibited by benzimidazoles 1) was reported to contribute to both chromosome congression and checkpoint function, yet the role of BUB1 catalytic activity in these processes remains a matter of debate.

To differentiate between catalytic and non-catalytic functions of BUB1 we compared phenotypes provoked by BUB1 protein depletion with specific BUB1 kinase inhibition using two novel small molecule inhibitors of BUB1, termed BAY-320 and BAY-524.

BAY-320 and BAY-524 were highly potent and selective ATP-competitive inhibitors of BUB1 kinase activity with IC50 values in the single digit nanomolar range (at 10 micromolar ATP concentration). By monitoring phosphorylation of Thr120 in histone H2A, we showed that both compounds acted as potent BUB1 kinase inhibitors both biochemically and in human cells. We found that BUB1 inhibition substantially altered the chromosomal association of Shugoshin and the chromosomal passenger complex without major effects on global Aurora B function. Consequently, inhibition of BUB1 kinase clearly impaired chromosome arm resolution but, in stark contrast to depletion of BUB1 protein, only had a minor effect on cell cycle and SAC function. Importantly, BAY-320 and BAY-524 treatment sensitized cells to low doses of paclitaxel, synergistically affecting chromosome segregation and cell proliferation.

These findings are highly relevant to both our understanding of BUB1 kinase function during mitosis and the prospects of BUB1 as a target of anti-cancer therapies. In this regard, BAY-320 and BAY-524 are first-in-class inhibitors of BUB1 kinase and their potential utility as anti-cancer agents is being explored.

Citation Format: Anna P. Baron, Conrad von Schubert, Fabien Cubizolles, Gerhard Siemeister, Marion Hitchcock, Anne Mengel, Jens Schröder, Amaury Fernández-Montalván, Martin Lange, Franz von Nussbaum, Dominik Mumberg, Erich Nigg. Probing mitotic functions of BUB1 kinase using the small molecule inhibitors BAY-320 and BAY-524. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2725.
Abstract 2726: WWP2 is required for normal cell cycle progression
Authors: Choi Byeong Hyeok , Che Xun , Chen Changyan , Lu Luo , Dai Wei
Published: July 2016

WWP2 is a ubiquitin E3 ligase belonging to the Nedd4-like family. Given that WWP2 target proteins including PTEN that are crucial for regulating cell proliferation or suppressing tumorigenesis, we have asked whether WWP2 plays a role in controlling cell cycle progression. Here we report that WWP2 is necessary for normal cell cycle progression as its silencing significantly reduces the cell proliferation rate. We have identified that an isoform of WWP2 (WWP2-V4) is highly expressed in the M phase of the cell cycle. Silencing of WWP2 accelerates the turnover of cyclin E, which is accompanied by increased levels of phospho-histone H3 (p-H3) and cyclin B. Moreover, silencing of WWP2 results in compromised phosphorylation of AktS473, a residue whose phosphorylation is tightly associated with the activation of the kinase. Combined, these results strongly suggest that WWP2 is an important component in regulating the Akt signaling cascade, as well as cell cycle progression.

Citation Format: Byeong Hyeok Choi, Xun Che, Changyan Chen, Luo Lu, Wei Dai. WWP2 is required for normal cell cycle progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2726.
Abstract 2728: In silico identification of a MAD2-interacting motif in MAD2 spliced isoforms suggest a functional interaction with the spindle assemble checkpoint in cancer
Authors: Ramirez-Otero Miguel , Lopez-Saavedra Alejandro , Andonegui Marco , Diaz-Chavez Jose , Herrera Luis Alonso
Published: July 2016

Abnormal chromosome segregation plays a key role in cancer development. MAD2 is a component of the spindle assembly checkpoint (SAC), a cell cycle control mechanism that ensures an accurate segregation of chromosomes during mitosis. Changes in MAD2 expression have been associated with chemo-resistance both to spindle inhibitors and to DNA damaging agents. Also, a previous study has shown that the exogenous expression of MAD2β, a splicing variant of MAD2, was associated with resistance to Adriamycin and Vincristine in gastric cell lines. Additionally, we have previously identified that exogenous overexpression of MAD2γ upon paclitaxel-induced SAC activation in the colorectal cancer cell HCT116, reduces drug-induced mitotic arrest. These findings suggested a possible structural interaction of MAD2 isoforms with SAC components. To determine possible structural interactions between MAD2 isoforms and key SAC components (i.e. MAD1 and CDC20), we performed an in silico analysis of MAD2 isoforms, Interestingly, we found that alternative splicing of MAD2 generates a premature stop codon and a frameshift in exon 4 in MAD2γ and MAD2β. This change generates a new C-terminal region in MAD2γ and MAD2β isoforms that comprise 16 amino acids, which are not present in the major isoform (MAD2α). We aligned this region with the amino acid sequence of CDC20 from various species and identified a MAD2-interacting motif (MIM). This finding suggests that MAD2 isoforms may interact with the active conformation of MAD2 (C-MAD2). Since MAD2 isoforms and CDC20 may compete for the same region in MAD2, we propose a new model whereby MAD2 isoforms inhibits SAC by interfering with C-MAD2/CDC20 formation. This model helps to explain previous results where MAD2 isoforms over expression seem to have an opposite role in SAC signaling.

Citation Format: Miguel Ramirez-Otero, Alejandro Lopez-Saavedra, Marco Andonegui, Jose Diaz-Chavez, Luis Alonso Herrera. In silico identification of a MAD2-interacting motif in MAD2 spliced isoforms suggest a functional interaction with the spindle assemble checkpoint in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2728.
Abstract 2734: Click colorimetric EdU proliferation and TUNEL assay: Conversion of click chemistry from fluorescent to colorimetric for use with bright field microscopy
Authors: Clarke Scott T. , Finnessy Carmen , Bosada Fernada M. , Armstrong Ben , O’Grady Michael
Published: July 2016

We present here the conversion of click chemistry fluorescence-based EdU proliferation assay and TUNEL apoptosis assay to use with bright field microscopy. Advantages of having a colorimetric signal are the retention of the click chemistry work flow while allowing for compatibility with standard H&E staining and protocols.

Proliferation using copper-catalyzed labeling of the thymidine analog EdU (ethynyl deoxyuridine) was introduced in 2007. This click-based assay has advantages over the traditional antibody based BrdU assay. The small molecule detection is simple, rapid and gives a bright signal useful for multiplexing. Additionally, recent improvements to the chemistry allow for GFP and R-PE compatibly with the EdU and TUNEL assays.

Several approaches for creating a colorimetric signal for EdU proliferation were attempted. The method presented here had the shortest protocol and uses a biotin-based click reaction followed by streptavidin -horseradish peroxidase (HRP) step. This two-step reaction creates an essentially covalent link between the EdU incorporated in the DNA and the enzyme reporter. Then standard HRP chromogens such as DAB were used to produce a signal marking the proliferating cells. Examples of proliferation using colorimetric EdU labeling in adult Zebrafish caudal fin, mouse cardiac, and rat intestine, mammary and uterine FFPE treated tissue are presented. Multiplexing with alkaline phosphatase for two color detection are also presented. Two color detection of EdU with BrdU or EdU with progesterone receptor (PR) are shown. Most HIER protocols are compatible with EdU detection. For EdU alone no HCl treatment is required. For EdU/BrdU double labeling the BrdU protocol for DNA revelation is used. Anti-BrdU mouse antibody clone MoBU-1 is used because of its lack of cross-reactivity with EdU.

Additionally, we present the click chemistry-based apoptotic TUNEL assay colorimetric detection using the same approach. Clickable biotin adapters are used to couple HRP to the ethynyl tails created by TdT enzyme extension at 3′ end of double stranded breaks. We provide examples of this late stage indicator of apoptosis visualized with DAB substrate and stained with standard H&E and other histological stains.

Citation Format: Scott T. Clarke, Carmen Finnessy, Fernada M. Bosada, Ben Armstrong, Michael O’Grady. Click colorimetric EdU proliferation and TUNEL assay: Conversion of click chemistry from fluorescent to colorimetric for use with bright field microscopy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2734.
Abstract 2745: A genetic screen to identify tumor suppressive DDR genes in breast cancer
Authors: Fagan-Solis Katerina D. , Gupta Gaorav P.
Published: July 2016

To maintain genomic integrity, cells utilize a DNA damage response (DDR) mechanism that 1) functions to repair damaged DNA efficiently and 2) commits cells to apoptosis if damage is irreparable. Failure of this mechanism results in genomic instability and cancer predisposition. Defects in DDR have been implicated in both sporadic and familial breast and ovarian cancer predisposition syndromes (e.g. BRCA1, BRCA2, and PALB2), although the specific pathway components that are disrupted have not been elucidated. In particular, an impaired DDR may contribute to the pathophysiology of triple-negative (ER-/PR-/HER2-) breast cancer (TNBC) due to the extent and complexity of the genomic instability that is observed. We identified the DNA damage sensing Mre11- Rad50-Nbs1 (MRN) complex as an essential mediator of the oncogene-induced DDR. Disrupting the MRN complex in mouse models promotes the development of genomically unstable, metastatic breast cancer. The clinical relevance of this pathway is highlighted by the observation that components of the Mre11 complex are perturbed in a significant fraction of human TNBCs, which correlates with clinical outcomes. However, lack of MRN suppression in a majority of TNBCs indicates that alternative DDR components may also be inactivated to promote breast cancer development. Our goal is to identify additional DDR genes that, when perturbed, promote the development of genomically unstable breast cancers. In order to accurately distinguish driver genes from bystander genes we have developed a platform to identify DDR genes that mediate oncogene-induced senescence in mammary epithelial cells using both shRNA and CRISPR pooled libraries. Analysis of newly identified tumor suppressor genes, compared to what is already known about Mre11 and p53 (positive controls), will enable us to determine causality of specific DDR mutations in breast cancer development and importantly, the genomic instability phenotype that is typical for TNBC. The insights gleaned from this study may ultimately provide the means to determine the optimal treatment regimen for each patient with TNBC, and to prevent over-treatment of patients where certain chemotherapies may not be effective.

Citation Format: Katerina D. Fagan-Solis, Gaorav P. Gupta. A genetic screen to identify tumor suppressive DDR genes in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2745.
Abstract 2752: Characterization of the ATM-SPOP pathway in prostate cancer tumorigenesis
Authors: Fried Joshua , Boohaker Rebecca , Zeng Qinghua , Zhang Wei , Xu Bo
Published: July 2016

The Speckle type Poz Protein (SPOP), an E3 ubiquitin ligase adaptor, has recently been identified as the gene that has the most common somatic point mutations in prostate cancer. SPOP mutations are associated with genomic alterations, indicating a role for SPOP in the maintenance of genome stability. We, and others, have recently demonstrated a critical role of SPOP in the DNA damage response (DDR), suggesting SPOP mutants may represent a subgroup of patients that have hyper sensitivity to DNA damaging therapies. However, how SPOP mutations might impact its function and their roles in the progress of prostate tumorigenesis remain to be extensively studied. Genomic studies have found that SPOP mutations are clustered within its substrate binding pocket. Of a particular interest, Serine119 is found to be frequently mutated to Asparagine (S119N). Using computational modeling, we found that Ser119 resides in the SBC-MATH binding interface and is in close contact with the non-polar residue of the SPOP-binding consensus motif. Therefore we hypothesized that phosphorylation of Serine 119 might directly affect substrate binding. First we characterized prostate cancer cells expressing this mutation and we found that they displayed a suboptimal DNA damage response with prolonged DNA repair. Using an in situ proximity ligation assay, we demonstrate that Serine 119 is essential for SPOP ionizing irradiation induced interaction with the Ataxia Telangiectasia Mutated kinase (ATM), a major hub protein in the DDR. With the evidence of SPOP being a phospho-protein, we further provide in vitro evidence that ATM phosphorylates SPOP on Serine 119 in response to DNA damage. Characterization of the functional significance of ATM-mediated SPOP phosphorylation indicates a wide range of downstream targets regulating cell cycle progression and DNA repair. Taken together, our data reveal a critical pathway linking ATM and SPOP in regulation of prostate cancer initiation and therapeutic responses to DNA damage. This also provides the first evidence of a pathophysiological relevant mutation linked to ATM phosphorylation in the DDR.

Citation Format: Joshua Fried, Rebecca Boohaker, Qinghua Zeng, Wei Zhang, Bo Xu. Characterization of the ATM-SPOP pathway in prostate cancer tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2752.
Abstract 2755: Regulation of 53BP1 by the structural nuclear protein NuMA
Authors: Salvador Moreno Naike
Published: July 2016

DNA double-strand breaks (DSBs) can cause genomic instability and cancer through point mutations and genomic translocations if they are not properly repaired. DSBs are repaired by two major pathways: homologous recombination (HR) or nonhomologous end joining (NHEJ). The cellular choice between these two pathways is critical to cell survival and is altered in cancers. 53BP1, which promotes NHEJ by blocking CtIP-dependent DNA resection, is a key protein mediating repair pathway choice. Here, we present a new negative regulation mechanism of 53BP1 that relies on the structural nuclear protein NuMA. Apart from its role in mitotic spindle assembly, NuMA influences chromatin organization during the interphase and modulates the chromatin response to DNA damage. We identified an interaction between NuMA and 53BP1 by proteomics analysis of a 53BP1 pull-down and confirmed this interaction by NuMA immunoprecipitation. We measured greater than 50% decreased 53BP1-NuMA binding in response to DSB, which suggest that, in the absence of DNA damage, NuMA may restrain 53BP1 diffusion. This hypothesis is supported by fluorescence correlation spectroscopy (FCS) measurements in cells expressing GFP-tagged 53BP1, as well as by microirradiation experiments in which NuMA overexpression or silencing inhibited or promoted 53BP1 recruitment at DNA damage sites, respectively. 53BP1 has an essential role in B cell class switch recombination (CSR) and mediates PARPi sensitivity in BRCA1-null cells. We will present how modulating NuMA expression affects both aspects of 53BP1 function. The results from our work will help to understand how 53BP1 is controlled, and shed light on the mechanisms regulating this modulator of PARPi sensitivity in the presence or absence of DNA damage.

Citation Format: Naike Salvador Moreno. Regulation of 53BP1 by the structural nuclear protein NuMA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2755.
Abstract 2757: Mcl-1 dictates DNA double-strand break repair pathway choice
Authors: Chen Guo , Xu Ke , Xie Maohua , Owonikoko Taofeek K. , Ramalingam Suresh S. , Doetsch Paul W. , Deng Xingming
Published: July 2016

DNA double-strand breaks (DSBs) are mainly repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways. The choice of pathway is a critical aspect of DSB repair that is poorly understood. We show that Mcl-1 acts as a functional switch in pathway choice between HR and NHEJ. Mcl-1 is cell cycle-regulated, with expression peaking in S/G2 phase via reduction of its ubiquitination when HR occurs. Mcl-1 depletion reduces HR and enhances NHEJ. Mcl-1 overexpression results in a net increase in HR over NHEJ. Mcl-1 promotes HR-dependent DSB repair following DNA replication stress, which contributes to maintenance of genomic integrity. Mcl-1 directly interacts with Ku via its BH1 and BH3 domains. This interaction is required for Mcl-1 to inhibit Ku-mediated NHEJ, and promote Mre11 complex-mediated DNA resection and HR-dependent DSB repair. Thus, Mcl-1, in addition to its antiapoptotic function, plays an unexpected role in directing DSB repair pathway choice by skewing the balance toward HR during cell cycle progression.

Citation Format: Guo Chen, Ke Xu, Maohua Xie, Taofeek K. Owonikoko, Suresh S. Ramalingam, Paul W. Doetsch, Xingming Deng. Mcl-1 dictates DNA double-strand break repair pathway choice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2757.
Abstract 2759: USP21 stabilizes the BRCA2/Rad51 complex to ensure cancer genome maintenance
Authors: Liu Jinping , Kruswick Alex J. , Dang Hien , Tran Andy D. , Wang Xin W. , Philipp Philipp
Published: July 2016

Cancer is a disease of defective genome maintenance. Paradoxically, however, tumor growth relies on efficient DNA repair to mitigate the detrimental impact of replication-associated DNA double-strand breaks (DSBs). Deleterious mutations in BRCA2, a tumor suppressor that promotes homologous recombination (HR), consequently result in increased susceptibility to genotoxic stress in rapidly dividing tumor cells. While these findings underscore the importance of tightly controlled BRCA2 function for tumor growth, little is known about the factors that regulate this process. Here, we establish the ubiquitin-specific protease USP21 as a positive regulator of BRCA2 stability. USP21 interacts with, deubiquitinates and stabilizes the BRCA2/Rad51 complex and promotes efficient Rad51 loading to DSBs. As a result, depletion of USP21 decreases HR efficiency, causes an increase in DNA damage load and impairs tumor cell survival. Importantly, BRCA2 overexpression partially restores the USP21-associated HR defect. Moreover, we show that USP21 is overexpressed in hepatocellular carcinoma (HCC), where it promotes BRCA2 stability and inversely correlates with patient survival. These findings demonstrate a novel deubiquitination mechanism to control BRCA2 levels and point to USP21 as a potential therapeutic target in BRCA2-proficient tumors.

Citation Format: Jinping Liu, Alex J. Kruswick, Hien Dang, Andy D. Tran, Xin W. Wang, Philipp Philipp. USP21 stabilizes the BRCA2/Rad51 complex to ensure cancer genome maintenance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2759.
Abstract 2760: Does a short open reading frame (sORF)-encoded protein participate in DNA repair
Authors: Hanakahi Les , Summerlin Matthew , Tang Yi-Chia , Heo Jinho
Published: July 2016

Non-homologous end joining (NHEJ) is one of the major mechanisms for rejoining of DNA double-strand breaks. Knowledge of the molecular mechanism of NHEJ will help us to understand how this process preserves genome integrity, and how NHEJ errors cause chromosomal rearrangements that lead to cancer. Several critical participants in the NHEJ pathway have been identified and some mechanistic steps have been characterized. At present, a significant gap in our understanding of NHEJ is how NHEJ factors assemble at DSBs. It is plausible that additional, as yet unidentified, factors contribute to NHEJ assembly. We have biochemical and biological data implicating a short open reading frame (sORF)-encoded protein in NHEJ. sORFs are found in all genomes. Ribosome profiling, mass spectroscopy and RNA sequencing have predicted the existence of numerous sORF-encoded proteins of <100 amino acids in size. While the functional significance of the majority of these small proteins is not clear, we recently discovered that a 69 amino acid protein encoded by chromosome 7 open-reading frame 49 (C7orf49) interacts with critical NHEJ factors in human cells, and stimulates NHEJ in cell-free extracts. Treatment of cells with the DSB-inducing agent etoposide increased C7orf49 expression and nuclear localization, and knockdown of C7orf49 sensitized cells to etoposide. C7orf49-deficient cells exhibited a mild proliferation defect and increased sensitivity to etoposide. Our data suggest that this small protein may play an important role in double-strand beak repair by NHEJ and highlights the potential biological relevance of sORF-encoded proteins in the DNA damage response and in DNA repair.

Citation Format: Les Hanakahi, Matthew Summerlin, Yi-Chia Tang, Jinho Heo. Does a short open reading frame (sORF)-encoded protein participate in DNA repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2760.