bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 623: Evaluation of a multiplex biomarker assay for the detection of prostate cancer cells in urine
Authors: Nickens Kristen , Ali Amina , Scoggin Tatiana , Tan Shyh-han , Ravindranath Lakshmi , McLeod David , Tacha David , Sesterhenn Isabell , Dobi Albert , Srivastava Shiv , Petrovics Gyorgy
Published: August 2015

Background and Objectives: Over one million men undergo prostate biopsies annually in the US, a majority of them due to elevated serum PSA. More than half of the biopsies turn out to be negative for prostate cancer (CaP). The limitations of PSA test as well as the biopsy procedure have prompted to develop more precise CaP detection assays in urine- (e.g. PCA3, TMPRSS2-ERG) or blood (e.g. PHI, 4K). Here we describe the development and evaluation of a Urine CaP Marker Panel (UCMP) assay for sensitive and reproducible detection of CaP cells in post-digital rectal examination (post-DRE) urine.

Methods: The cellular content of the post-DRE urine, was captured on translucent membrane for direct evaluation by microscopy and immunocytochemistry. The membranes placed on Cytoclear microscope slides were used for characterization of prostate epithelial cell by PSA and Prostein or tumor cells by ERG and AMACR protein markers. Immunostained cells were analyzed for quantitative and qualitative features and correlated with biopsy positive and negative status for malignancy.

Results: Assay was optimized for single cell capture sensitivity and downstream evaluations by spiking known number of cells from established prostate cancer cell lines, LNCaP and VCaP, into cleared control urine. The cells captured from the post-DRE urine of subjects, obtained prior to biopsy procedure, were co-stained for ERG, AMACR, Prostein and PSA, rendering a whole cell based analysis and characterization. A feasibility cohort of 63 post-DRE urine specimens was assessed. Comparison of the UCMP results with the blinded biopsy results showed an assay sensitivity of 64% (16 of 25) and a specificity of 68.8% (22 of 32) for CaP detection by biopsy.

Conclusion: This pilot study assessing a minimally invasive prostate cancer detection assay with single cell sensitivity cell-capture and characterization from the post-DRE urine holds promise for further development of this novel assay platform.

Citation Format: Kristen Nickens, Amina Ali, Tatiana Scoggin, Shyh-han Tan, Lakshmi Ravindranath, David McLeod, David Tacha, Isabell Sesterhenn, Albert Dobi, Shiv Srivastava, Gyorgy Petrovics. Evaluation of a multiplex biomarker assay for the detection of prostate cancer cells in urine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 623. doi:10.1158/1538-7445.AM2015-623
Abstract 63: Alview (ALignment VIEWer): A software tool to visualize next generation sequencing (NGS) data
Authors: Meerzaman Daoud , Finney Richard , Chen Qing-Rong , Nguyen Cu , Hsu Chih Hao , Dunn Barbra
Published: August 2015

Widespread use of NGS has led to the increasing sizes of high throughput sequencing data of various types from different platforms. Review of alignment data by experienced genomics researchers is an important quality control element in this process. Key to this activity is visual inspection of the sequencing data in order to eliminate false positives and document the likelihood of true positives. Furthermore, such manual, or visual, examination of data must occur at extremely rapid speeds.

Alview (for “ALignment VIEWer”) is a compiled to native architecture software tool for visualizing the alignment of sequencing data. The inputs data are in the SAM/BAM format short read sequences that are aligned to a reference genome sequence. Outputs are images’ representing these short reads aligned to the reference genome. Alview is written in portable C with optional Graphical User Interface (GUI) code written in C,C++ or Objective C. Alview can be compiled to run in three different ways: (1) as a webserver, (2) as a command line tool, or (3) Alview is executable as a native graphical user interface. Alview is compatible with Microsoft Windows, Linux, and Apple OS X. Our new tool saves time and addresses specific regions of interest (for example, regions containing known or novel mutations or SNPS) and peruses these regions in the manner of a “slideshow”, enabling the scanning of a large number of samples in a short time

Citation Format: Daoud Meerzaman, Richard Finney, Qing-Rong Chen, Cu Nguyen, Chih Hao Hsu, Barbra Dunn. Alview (ALignment VIEWer): A software tool to visualize next generation sequencing (NGS) data. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 63. doi:10.1158/1538-7445.AM2015-63
Abstract 636: Next-generation site-specific antibody-drug conjugates using the SMARTagTM technology platform
Authors: Kudirka Romas
Published: August 2015

The two clinically-approved antibody-drug conjugates, AdcetrisTM and KadcylaTM, demonstrate that targeting cytotoxic payloads directly to tumors is an effective clinical approach. However, at the molecular level, these therapeutics are composed of heterogeneous mixtures resulting from the nonselective ligation of cytotoxic payload to cysteine and lysine residues, respectively. The nonselective ligation approaches used to generate these two ADCs render it difficult to optimize their biological, physical, and pharmacological properties. Redwood Bioscience creates site-selective, stable linkages that allow us to design ADCs with enhanced efficacy, safety, and pharmacokinetics. Additionally, we are able to control payload stoichiometry and placement. Redwood Bioscience creates homogeneous ADCs using the SMARTagTM technology, in which a genetically encoded 5 amino acid “aldehyde-tag” sequence contains a cysteine that is cotranslationally converted to formylglycine via an enzymatic transformation. This method affords control over the placement of the reactive formylglycine aldehyde side chain, which can then be ligated using carbonyl bioconjugation methods. Here, we will present our novel protein modification platform—including our new conjugation chemistries and linker libraries—and its application to ADC generation. We will address SMARTagTM ADC safety, pharmacokinetics, and efficacy in preclinical tumor models, and discuss how these parameters are impacted by linker selection and payload placement on the antibody.

Citation Format: Romas Kudirka. Next-generation site-specific antibody-drug conjugates using the SMARTagTM technology platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 636. doi:10.1158/1538-7445.AM2015-636
Abstract 64: Targeting immune checkpoints: using a big data approach for their identification, prioritization and application
Authors: Kaw Sheetal , Upmanyu Sanatan , Sharma Himani , Nandabalan Krishnan
Published: August 2015

Tumor cells have long been known to successfully evade immune surveillance mechanisms.In order to curtail the tumor progression as well as to develop effective therapeutic anti-tumor strategies, key immune regulators,or so-called immune checkpoints, havebeen recognizedas playing an important role. To prevent excess tissue damage,immune checkpoints that refer to a number of inhibitory players involved in immune responses,are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses.Therapies targeting such immune checkpoints such as CTLA-4 and PD-1 have shown great clinical successas they boost the already existing immune response working against the tumorprogression. To identify novel immune checkpoints it becomes crucial to comprehensively analyze the wealth of existing knowledge regarding key signaling pathways, theircross-talk with immune regulators, protein expression profiles in tumor tissues, and patient outcomes data. These data silos require a big data analytics approach to successfully hypothesize, query and derive meaningful conclusions from this data. With a proprietary big data analytics platform PharmGPS™, we have identified a number of immune checkpoints that have clear therapeutic potential for solid tumor regenesis. First, second and third degree associations between tumorigenic pathways and immune checkpoints were identified and preliminary findings will be presented.

Citation Format: Sheetal Kaw, Sanatan Upmanyu, Himani Sharma, Krishnan Nandabalan. Targeting immune checkpoints: using a big data approach for their identification, prioritization and application. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 64. doi:10.1158/1538-7445.AM2015-64
Abstract 640: Probing the tumorigenic properties of two pore potassium channels using inhibitory anti-KCNK9 mAbs
Authors: Sun Han , Luo Liqun , Lal Bachchu , Laterra John , Li Min
Published: August 2015

Ion channels are important portals that facilitate ionic passage across membranes in all organisms. KCNK9, a member of the family of two-pore domain potassium K+ channels, mediates “leak” currents critical to maintaining cell resting membrane potentials. Since its identification, KCNK9 has been implicated in various human disorders including cancer. KCNK9 overexpression has been observed in melanoma, colorectal, breast and lung tumors and KCNK9 has been found to promote tumor survival and growth, by increasing resistance to hypoxia and apoptosis. However, KCNK9's underlying pathophysiologic mechanisms have been elusive due to the absence of specific inhibitors. Here, we describe the development of novel monoclonal antibodies directed against the KCNK9 extracellular domain and their molecular/cellular effects on KCNK9-expressing cells.

Murine monoclonal IgG1 antibodies were raised against recombinant KCNK9 extracellular domains expressed in a mammalian system. 40 monoclonal antibodies were generated and demonstrated channel subtype-specific recognition of human KCNK9. Among them, 1 antibody designated Y4-mAb displayed sub-nanomolar binding affinity and inhibited KCNK9 channel conductance by up to 30% when tested in a fluorescent ion-influx assay. Through confocal microscopic analysis, channel inhibition was found to result from antibody-induced internalization and endocytosis of cell-surface channels. Y4-mAb induced up to 30∼40% reduction in cell viability and increased cell death in BEN non-small cell lung carcinoma cells and BT-549 and 410.4 breast cancer cells. In addition to “direct cell killing”, this antibody activated complement, resulting in complement-dependent cancer cell cytotoxicity in vitro. Initial experiments in vivo showed that systemic Y4-mAb inhibited the capacity of BEN non-small cell lung carcinoma cells to propagate subcutaneous tumor xenografts in nude mice (p<0.05).

In conclusion, we have generated a high affinity KCNK9-specific monoclonal antibody that perturbs KCNK9 function and inhibits tumor cell survival and xenograft formation. Anti-KCNK9 antibodies have the potential to be valuable research tools and may lead to novel therapeutics. Moreover, our strategies should be generally applicable to studying the functions of other ion channels in cancer.

Citation Format: Han Sun, Liqun Luo, Bachchu Lal, John Laterra, Min Li. Probing the tumorigenic properties of two pore potassium channels using inhibitory anti-KCNK9 mAbs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 640. doi:10.1158/1538-7445.AM2015-640
Abstract 644: Impact of conjugation site on pharmacokinetics and off-target toxicity of site-specific antibody drug conjugates
Authors: Ma Dangshe , Jin Fang , Barletta Frank , Hu George , Tumey Nathan , Zhang Haige , He Tao , Sousa Eric , Charti Manoj , Khadke Kiran , Lucas Judy , Ferguson Darren , Brown Christoper , Ma Weijun , Gatto Scott , Brady William , Graziani Edmund , Gerber Hans-Peter , Sapra Puja , Tchistikova Lioudmila
Published: August 2015

Site-specific conjugation of toxic payload to antibody yields homogeneous ADCs resulting in improved therapeutic index over conventional conjugation. To understand the role of conjugation site on pharmacokinetics, off-target toxicity, cysteine(cys) was engineered into a humanized anti-IL13Rα2 antibody, hAB08, at various sites within the IgG1/k constant domains. Single cys mutants L443C and Q347C with DAR 2, and double cys mutants K392C+L443C, L443C/kK183C and Q347C/kK183C with DAR 4 were conjugated using auristatin 0101 via maleimidocapronic - valine-citruline-p-aminobenzyloxycarbonyl (vc, cleavable linker). All cys mutants and their conjugates maintained their binding properties compared to parental hAb08. Cysteine substitution sites were selected based on conjugation efficiency and in vitro stability. In vivo efficacy was evaluated in mouse xenograph tumor models. Pharmacokinetics (PK) and off target toxicity (TOX) were assessed in both mouse and rat in vivo studies. Our data demonstrate that that all site specific conjugates show improved in vitro stability, comparable or better in vivo efficacy in mice xenograph models and in vivo stability (ratio of ADC-AUC/Ab-AUC) compared to conventional conjugates. However PK and off-target toxicity of antibody drug conjugates had been significantly impacted by conjugation site and animal species. L443 mutants show higher ADC-AUC, lower clearance and longer half life than Q347 mutants in mice while a reverse TK behavior was observed in rat. All site specific conjugates show higher dose tolerance and less off-target toxicity relative to conventional conjugates in rat. This off-target toxicity improvement depends on sites of conjugation. The L443 mutants exhibited a better off-target TOX profile relative to Q347 mutants. Taken together, site-specific conjugation improved in vivo efficacy and off-target toxicity relative to conventionally conjugated ADC. PK differences and off-target toxicity improvements depend on conjugation sites and animal species.

Note: This abstract was not presented at the meeting.

Citation Format: Dangshe Ma, Fang Jin, Frank Barletta, George Hu, Nathan Tumey, Haige Zhang, Tao He, Eric Sousa, Manoj Charti, Kiran Khadke, Judy Lucas, Darren Ferguson, Christoper Brown, Weijun Ma, Scott Gatto, William Brady, Edmund Graziani, Hans-Peter Gerber, Puja Sapra, Lioudmila Tchistikova. Impact of conjugation site on pharmacokinetics and off-target toxicity of site-specific antibody drug conjugates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 644. doi:10.1158/1538-7445.AM2015-644
Abstract 649: Supramolecular assembly of antibody-drug conjugates using CORDLink platform for targeted drug delivery
Authors: Gupta Nimish , Kancharla Johny , Kaushik Shelly , Hossain Samad , Sarkar Arindam , Sengupta Aniruddha , Roy Monideepa , Sengupta Shiladitya
Published: August 2015

Antibody-drug conjugates (ADCs) have emerged as a promising class of cancer therapeutics for targeted drug delivery. However, the synthesis of novel linkers for generating stable ADCs remains a key goal in the development of effective ADCs. Here we describe a unique linker (CORDLink) platform technology based on coordination chemistry that maintains the integrity of the antibody without impacting binding to the target. Using an anti-HER2 monoclonal antibody and camptothecin, as examples of the targeting antibody and a highly cytotoxic payload respectively, we demonstrate that the linker enables the formation of a stable ADC via supramolecular coordinate bond. The robustness of our process and consistency was characterized through a variety of analytical tools, such as SE-HPLC, CEX-HPLC and HI-HPLC, while peptide mapping was done to elucidate the exact site of attachment. The ADCs were found to remain intact in media and serum using a fluorescence-based RP-HPLC method. The anti-HER2 antibody recognizes HER2 receptors on cancer cells inducing internalization of the conjugate. The ADC showed significant cytotoxicity in cells expressing HER2 receptor but not in HER2 negative cell lines with IC50s in the nM range. We have established robust characterization methods to validate and confirm the integrity of the prototype ADCs in vitro. Studies are underway to further evaluate these molecules in vivo. The CORDLink technology offers a highly versatile platform that can be leveraged to deliver a broad range of payloads to selected cells using different targeting molecules.

Citation Format: Nimish Gupta, Johny Kancharla, Shelly Kaushik, Samad Hossain, Arindam Sarkar, Aniruddha Sengupta, Monideepa Roy, Shiladitya Sengupta. Supramolecular assembly of antibody-drug conjugates using CORDLink platform for targeted drug delivery. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 649. doi:10.1158/1538-7445.AM2015-649
Abstract 65: An integrated bioinformatics approach for identifying patient-specific gene networks and novel therapeutic targets in glioblastoma
Authors: Stathias Vasileios , Pastori Chiara , Komotar Ricardo , Zhang Ming , Schürer Stephan , Clarke Jennifer , Ayad Nagi G.
Published: August 2015

The purpose of our approach is to identify highly relevant and patient specific gene networks from next generation sequencing data and use the resulting gene combinations to identify potential novel therapeutic targets.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant brain tumor. Despite medical advances in the field, the median survival time is still below 2 years since recurrence is nearly universal. Thus, the discovery of novel and specific molecular targets is needed.

As with all cancers, GBM arises due to complex alterations in a patient's genome. Identifying patient-specific differentially expressed genes (DEGs) though, can impose many difficulties given the discordance of different analysis methods when the sample size is low. We used the data on the large number of GBM patients within TCGA (The Cancer Genome Atlas) as filter for identifying variations in expression. With the help of our algorithm, we produced enriched single patient RNAseq DEG lists and we calculated a hypergeometric probability and a correlation coefficient for every gene pair on these lists. By using the most significant of these pairs, we generated gene association networks. To further validate the biological relevance of the gene pairs, we looked into the presence of these networks in other types of cancer and searched for published experimental data supporting the connections of our networks. In order to assess the therapeutic potential of these genes, we used complimentary experiments involving GBM cell lines recorded through the LINCS (Library of Integrated Network-based Cell Signatures) database.

This study identifies a putative workflow for uncovering differentially expressed patient specific genes and gene networks for GBM, and capitalizes on the LINCS database to assess the potential of novel therapeutic targets.

Citation Format: Vasileios Stathias, Chiara Pastori, Ricardo Komotar, Ming Zhang, Stephan Schürer, Jennifer Clarke, Nagi G. Ayad. An integrated bioinformatics approach for identifying patient-specific gene networks and novel therapeutic targets in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 65. doi:10.1158/1538-7445.AM2015-65
Abstract 650: A high-throughput conjugation strategy for the selection of THIOMAB™ antibodies with desired properties for antibody-drug conjugation
Authors: Ohri Rachana V. , Bhakta Sunil , Raab Helga , Vandlen Richard , Junutula Jagath , Erickson Hans
Published: August 2015

THIOMAB™ antibody technology utilizes antibody engineering to replace endogenous amino acids of an antibody with cysteine to allow for site-selective conjugation. The technology enables the exploration of different drug attachment sites on the antibody thereby offering the potential to select antibody-drug conjugates (ADCs) with unique properties. Having a large number of antibody sites from which to choose may be desired for some applications. Many antibody sites are not suitable due to limited solvent accessibility or because drug attachment alters the native antibody properties. To identify those antibody sites most amenable to drug conjugation, we prepared 648 THIOMAB™ antibody variants of the anti-HER 2 antibody 4D5 by sequentially mutating every amino acid in the light and heavy chains to cysteine (excluding the endogenous cysteines). Each THIOMAB™ antibody variant was then conjugated to maleimido caproyl valine citrulline PABA MMAE and pyridyl disulfide MMAE in separate reactions. A high throughput on-bead Protein A conjugation and purification method was developed using a 96 well plate format. Complete reduction of the engineered cysteines and reoxidation of the endogenous cysteines, and drug to antibody ratios (DAR) were all determined by LC/MS. The concentration and aggregation status of each ADC were determined using automated liquid chromatography methods. Nearly 40% of the THIOMAB™ antibodies were found to be suitable for conjugation with DAR >1 and low aggregation levels. The other 60% failed due to a variety of factors including low drug incorporation, high aggregation levels, and incomplete re-oxidation of the endogenous cysteines. We found similar results with the maleimide and pyridyl disulfide derivatives suggesting our findings may be applicable to other auristatin derivatives with different sulfhydryl reactive chemistries. In summary, we have developed a high throughput conjugation strategy for THIOMAB™ antibodies and used it to identify the antibody sites most suitable for conjugation to auristatin derivatives.

Citation Format: Rachana V. Ohri, Sunil Bhakta, Helga Raab, Richard Vandlen, Jagath Junutula, Hans Erickson. A high-throughput conjugation strategy for the selection of THIOMAB™ antibodies with desired properties for antibody-drug conjugation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 650. doi:10.1158/1538-7445.AM2015-650
Abstract 654: PG-101 in combination with docetaxel or Herceptin improves breast cancer survival
Authors: Wadehra Madhuri , Kiyohara Meagan , Dillard Christen , Ashki Negin , Qin Christie
Published: August 2015

Background: Breast cancer is a heterogeneous disease with several subtypes which dictate which course of treatment each patient will receive. However, resistance remains a significant challenge. In triple negative breast cancer (TNBC), an aggressive histological subtype with limited treatment options and very poor prognosis, resistance to current standard therapies such as taxanes severely limits the available options for previously treated patients. This problem also occurs with targeted therapies. Trastuzumab is the standard of care for HER2+ tumors. However, de novo or acquired Trastuzumab resistance is a major limitation for its clinical efficacy. Thus, there remains an urgent unmet need for improved targeted agents for this patient population.

Methods: To this end, we define another marker epithelial membrane protein-2 (EMP2) as a novel target for Trastuzumab and taxane resistant tumors. EMP2 expression was determined using standard immunohistochemistry. We have recently developed a novel IgG1 monoclonal antibody to EMP2 termed PG-101. Acquired Trastuzumab and docetaxel resistant tumors were treated with PG-101 using human xenograft models.

Results: EMP2 is an oncogenic protein whose expression has been shown to correlate with tumor progression as well as the formation of breast cancer stem cells. In this study, we present evidence that EMP2 is highly expressed in acquired resistant tumor cells both in culture as well as in patient samples. We also tested the efficacy of PG-101 in combination with docetaxel, and we have evidence that the combination significantly improves tumor-free survival. Moreover, preliminary data also strongly suggests that PG-101 can be used as an adjuvant therapy for docetaxel and Trastuzumab resistant tumors whereby PG-101 reduced both tumor load in both models in vivo.

Conclusions: Given that EMP2 is also overexpressed in endometrial and ovarian cancers, our data collectively suggest that PG-101 therapy has the potential to revolutionize the outcomes for women with cancer.

Citation Format: Madhuri Wadehra, Meagan Kiyohara, Christen Dillard, Negin Ashki, Christie Qin. PG-101 in combination with docetaxel or Herceptin improves breast cancer survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 654. doi:10.1158/1538-7445.AM2015-654
Abstract 655: The next generation of targeted toxins: A novel deimmunized sarcin ribotoxin fused with an EphA2 Abdurin binder
Authors: Gehlsen Kurt R. , Demartis Anna , Jones Tim
Published: August 2015

Abdurins are a novel antibody-like scaffold derived from the engineering of a single CH2 domain (CH2D) of IgG. The prolonged serum half-life of Abdurins, the result of retained FcRn binding, is 10-20 times longer than the serum half-life of any other protein scaffold of comparable molecular weight. Here we present data on the isolation and characterization of high affinity ABDURIN binders to the cancer target EphA2. α-Sarcin is a small (17 kDa) fungal ribonuclease produced by Aspergillus giganteus. Sarcin functions by catalytically cleaving a single phosphodiester bond in the sarcin-ricin loop, a well-defined RNA motif within the rRNA scaffold of the large ribosomal subunit. This cleavage makes the ribosome unrecognizable to elongation factors and hence, blocks protein synthesis. We have deimmunized α-sarcin (Sarcin-DI) and made several variants lacking T cell epitopes while retaining cytotoxic activity. This novel Abdurin EphA2 binder fusion with Sarcin-DI creates a new generation of targeted toxins with a longer half-life, low immunogenicity and potent tumor cell killing.

Citation Format: Kurt R. Gehlsen, Anna Demartis, Tim Jones. The next generation of targeted toxins: A novel deimmunized sarcin ribotoxin fused with an EphA2 Abdurin binder. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 655. doi:10.1158/1538-7445.AM2015-655
Abstract 657: Microtubule stabilizers inhibit the cellular transport and signaling of EGFR in breast cancer cells
Authors: Rohena Cristina Coral , Dybdal-Hargreaves Nicholas F. , Mooberry Susan L.
Published: August 2015

Breast cancer is the second most diagnosed cancer in women and it is estimated that approximately 40,000 women die every year from this disease in this country alone. The overexpression of EGFR in breast cancer is associated with drug resistance and an aggressive nature resulting in an overall poor outcome. The EGFR signaling pathway requires internalization of the receptor, endosomal translocation and in some cases nuclear accumulation of the receptor. These processes are dependent on functional microtubules. Microtubule targeting agents (MTAs) have been used with great success in the treatment of breast cancer alone and in combination with other agents. MTAs impair microtubule dynamics, which results in the inhibition of mitosis. Microtubules are additionally important in cellular signaling events including cellular transport of the EGFR. The effects of the microtubule stabilizer taccalonolide AJ on EGFR transport and activation in breast cancer cells were compared to the effects of paclitaxel. Our results show that microtubule stabilization interrupts the cellular trafficking of EGFR, leading to the retention of the receptor in the cell periphery.. Taccalonolide AJ and paclitaxel impaired the association of EGFR with the early endosome and delayed its association with the lysosome. This disruption in cellular transport also led to decreased phosphorylation of EGFR downstream targets AKT, ERK1/2 and STAT3, with taccalonolide AJ being the more effective inhibitor. Our studies show that microtubule stabilizers also prevent the association of EGFR with the Golgi instead leading to the fragmentation of this organelle. Paclitaxel caused a higher degree of Golgi fragmentation and it was observed in nearly all cells. The inhibition of EGFR localization to the Golgi caused by taccalonolide AJ and paclitaxel suggests that microtubule stabilization interferes with the trafficking of EGFR to the nucleus. This was confirmed by our results showing that microtubule stabilization impairs the nuclear transport of EGFR in breast cancer cells. Not surprisingly, in this case, paclitaxel was much more effective at preventing this nuclear accumulation. These studies highlight the utility and versatility of microtubule stabilizers and suggest the potential for combination treatments with microtubule stabilizers and small molecule inhibitors of EGFR and EGFR dependent pathways in breast cancer.

Citation Format: Cristina Coral Rohena, Nicholas F. Dybdal-Hargreaves, Susan L. Mooberry. Microtubule stabilizers inhibit the cellular transport and signaling of EGFR in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 657. doi:10.1158/1538-7445.AM2015-657
Abstract 658: Phosphorylated HER3 levels associated with trastuzumab resistance in HER2 gene amplified uterine serous carcinoma xenograft tumors
Authors: Hernandez Silvia F. , DiGloria Celeste , Groeneweg Jolijn , Borger Darrell , Foster Rosemary , Rueda Bo , Growdon Whitfield
Published: August 2015

Uterine serous carcinoma (USC) is an aggressive subtype of endometrial cancer that commonly harbors HER2 gene amplification. Clinical trial has demonstrated that USC is impervious to trastuzumab therapy, though the mechanism is poorly understood. Since HER3 mediated signaling has been implicated in trastuzumab resistance in breast cancer, we sought to understand the relevance of HER3 activation in USC xenografts derived from the HER2 gene amplified (HER2:Chr 17 > 15) non-immortalized USC cell line ARK2. Cohorts of mice harboring xenografts derived from ARK2 were treated with either vehicle, trastuzumab (10 mg/kg IP BIW), lapatinib (150 mg/kg QD oral gavage) or the combination of trastuzumab and lapatinib for 21 days. Acute and chronic post treatment tumor samples were assessed for downstream signaling alterations. Single agent trastuzumab had no impact on xenograft growth compared to vehicle. Lapatinib alone resulted in significant tumorstatic effects (p < 0.01) and dual therapy with trastuzumab and lapatinib induced synergistic activity that significantly decreased tumor volume compared to all other arms (p < 0.01). Single agent trastuzumab was associated with rapid elevation in pHER3 levels with unchanged pAKT and pERK expression compared to vehicle. These elevated pHER3 levels were similarly elevated after the 21 day treatment. Anti-tumor activity observed in the lapatinib and dual lapatinib/trastuzumab arm was not associated with any alterations in pHER3 levels following 21 day treatment, though acute elevations in pHER3 were noted in the dual HER2 blockade arm at 24 hours after treatment. In conclusion, trastuzumab alone failed to impact USC xenograft growth and this innate resistance was associated with an elevation in pHER3 levels that was still evident in xenografts at the end of the treatment period. Unlike single agent trastuzumab, treatment arms that utilized lapatinib demonstrated significant anti-tumor activity with no increase in pHER3 above vehicle over the course of treatment. These data highlight HER3 activation as a possible trastuzumab resistance mechanism in USC that can be abrogated through the addition of lapatinib.

Citation Format: Silvia F. Hernandez, Celeste DiGloria, Jolijn Groeneweg, Darrell Borger, Rosemary Foster, Bo Rueda, Whitfield Growdon. Phosphorylated HER3 levels associated with trastuzumab resistance in HER2 gene amplified uterine serous carcinoma xenograft tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 658. doi:10.1158/1538-7445.AM2015-658
Abstract 660: Polyisoprenylated cysteinyl amide inhibitors exhibit anti-tumor effects in human breast cancer cell lines
Authors: Salako Olufisayo O. , Poku Rosemary A. , Nkembo Augustine T. , Mazu Typhon , Flores-Rozas Hernan , Lamango Nazarius S.
Published: August 2015

PURPOSE: Triple negative breast cancer (TNBC) is the most aggressive and fatal form of breast cancer. TNBC has no effective therapies since it is not driven by the estrogen (ER), progesterone (PR), and human epidermal growth factor type 2 (Her2/Neu) receptors for which there are targeted drugs. Human epidermal growth factor receptors (EGFR) are overexpressed in 92% of TNBC cases, however, anti-EGFR therapies are ineffective. Monomeric G-proteins that regulate cell proliferation, angiogenesis, migration and apoptosis operate downstream of EGFR and other signal transduction pathways. Polyisoprenylated methylated protein methyl esterase (PMPMEase) metabolizes monomeric G-proteins, allowing them to function properly. PMPMEase is overexpressed in many cancers and may serve as a biomarker and target for cancer therapy. We have synthesized polyisoprenylated cysteinyl amide inhibitors that may inhibit PMPMEase and/or disrupt polyisoprenylated protein function. We hypothesize that the PCAIs will promote anti-tumorigenic effects, inducing apoptosis, while inhibiting cell survival, proliferation, migration, and invasion. The purpose of this study was to test the effectiveness of PCAIs against the biological hallmarks of TNBC.

METHODS: Four TNBC cell lines were treated with the PCAIs and analyzed for their effects on cell survival, viability, F-actin organization, cell migration and apoptosis.

RESULTS: Treatment of TNBC cells with the PCAIs resulted in a concentration-dependent death of the TNBC cells with EC50 values ranging from 2.13 to 2.78 μM for NSL-BA-040 and NSL-BA-055 in media containing 5% FBS. AO/EB staining revealed early apoptosis at 1 μM. NSL-RD-036 inhibited MDA-MB-231 cell migration by 71% (163±10.7 cells in control versus 47±6.9 in treated (±SEM, N = 3). PCAIs induced complete disruption of F-actin filament organization at 2 μM in MDA-MB-468 cells.

CONCLUSION: The PCAIs’ ability to induce apoptosis, and inhibit cell migration by disrupting F-actin filaments suggest that their potential therapeutic value could involve the inhibition of TNBC tumor growth, metastasis, and angiogenesis, which are all essential hallmarks for breast cancer progression. The PCAIs may ultimately fill the targeted therapy void in TNBC therapy.

Citation Format: Olufisayo O. Salako, Rosemary A. Poku, Augustine T. Nkembo, Typhon Mazu, Hernan Flores-Rozas, Nazarius S. Lamango. Polyisoprenylated cysteinyl amide inhibitors exhibit anti-tumor effects in human breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 660. doi:10.1158/1538-7445.AM2015-660
Abstract 662: USP8 modulates ubiquitination of LRIG1 for Met degradation
Authors: Lee Ji Min , Kim Bogyou , Kim Kyung-Ah
Published: August 2015

The Met receptor tyrosine kinase is an attractive target for cancer therapy as it promotes invasive tumor growth. SAIT301 is a novel anti-Met antibody, which induces LRIG1-mediated Met degradation and inhibits tumor growth. However, detailed downstream mechanism by which LRIG1 mediates target protein down-regulation is unknown. In the present study, we discovered that SAIT301 induces ubiquitination of LRIG1, which in turn promotes recruitment of Met and LRIG1 complex to the lysosome through its interaction with Hrs, resulting in concomitant degradation of both LRIG1 and Met. We also identified USP8 as a LRIG1-specific deubiquitinating enzyme, reporting the interaction between USP8 and LRIG1 for the first time. SAIT301 triggers degradation of LRIG1 by inhibiting the interaction of LRIG1 and USP8, which regulates ubiquitin modification and stability of LRIG1. In summary, SAIT301, a Met targeting antibody, employs ubiquitination of LRIG1 for its highly effective Met degradation. This unique feature of SAIT301 enables it to function as a fully antagonistic antibody without Met activation. We found that USP8 is involved in deubiquitination of LRIG1, influencing the efficiency of Met degradation. The relation of Met, LRIG1 and USP8 strongly supports the potential clinical benefit of a combination treatment of a USP8 inhibitor and a Met inhibitor, such as SAIT301.

Citation Format: Ji Min Lee, Bogyou Kim, Kyung-Ah Kim. USP8 modulates ubiquitination of LRIG1 for Met degradation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 662. doi:10.1158/1538-7445.AM2015-662
Abstract 663: Targeting autophagy potentiates the anti-tumoral action of crizotinib in ALK-positive anaplastic large cell lymphoma
Authors: MITOU Géraldine , FRENTZEL Julie , LAMANT Laurence , MEGGETTO Fabienne , ESPINOS Estelle , CODOGNO Patrice , BROUSSET Pierre , GIURIATO Sylvie
Published: August 2015

ALK (Anaplastic Lymphoma Kinase)-positive Anaplastic Large Cell Lymphoma (ALCL) occurs predominantly in children and young adults. Their chemotherapeutic treatment leads to a 5-year overall survival amounted to 70-80%. The tumor relapses are often very aggressive and lethal and their underlying mechanisms are unknown. Therefore, there is still a need to improve current therapy. Crizotinib is the most advanced ALK tyrosine kinase inhibitor already used in clinics for ALK-associated lung cancers. However, mechanisms of escape to crizotinib have been reported in cell lines and patients submitted to continuous crizotinib treatment. Thus, its use in frontline treatment for ALCL is hampered by the emergence of resistance. As autophagy has been proposed as a cell survival mechanism potentially involved in the acquisition of resistance to tyrosine kinase inhibitor, we investigated here whether autophagy was activated during ALCL treatment. We demonstrated in ALCL that autophagy is induced upon ALK inactivation, as a pro-survival response. We found that different ALK inhibition approaches (crizotinib or ALK-targeting siRNA) combined with autophagy inhibition (chloroquine, 3-methyladenine or ATG7-targeting siRNA) compromised cell survival and cell growth. Altogether, our results suggest that crizotinib and chloroquine (two drugs already used in clinics) co-treatment could be beneficial for ALK-positive ALCL patients.

Citation Format: Géraldine MITOU, Julie FRENTZEL, Laurence LAMANT, Fabienne MEGGETTO, Estelle ESPINOS, Patrice CODOGNO, Pierre BROUSSET, Sylvie GIURIATO. Targeting autophagy potentiates the anti-tumoral action of crizotinib in ALK-positive anaplastic large cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 663. doi:10.1158/1538-7445.AM2015-663
Abstract 665: Inhibition of mitochondrial complex I enhances the therapeutic benefit of BRAF inhibition in mutant melanoma cell lines
Authors: Schoeckel Laura , Bitschar Katharina , Héroult Mélanie , Kopitz Charlotte , Hägebarth Andrea
Published: August 2015

Metabolic reprogramming is considered to be one of the major hallmarks of cancer. Selective inhibitors of mutant BRAFV600E, are a widely used targeted therapy for melanoma. Unfortunately, acquired drug resistance, which commonly occurs, represents a major limitation to clinical efficacy. Recent studies have highlighted the role of BRAF inhibition in promoting oxidative phosphorylation (OXPHOS), over glycolysis, in BRAFV600E melanomas, a switch commonly seen in conjunction with development of drug resistance. This metabolic switch occurs at the transcriptional level, indicated by the up-regulation of PGC1α through the lineage-specific transcription factor MITF. Based on these data, we propose that increased OXPHOS is required for melanoma cells to acquire resistance against the BRAF inhibition, which supports our investigation of the therapeutic benefit of combining BRAF and OXPHOS inhibitors. For the latter, respiratory Complex I, an essential enzyme in the mitochondrial respiratory chain, is a logical target for OXPHOS inhibition.

We investigated the molecular, metabolic and bioenergetic effects of a selective mutant BRAF inhibitor (Vemurafenib/PLX4032) and BAY 87-2243, a small molecule inhibiting respiratory Complex I, in vitro using various BRAF-mutant melanoma cell lines. First, our study revealed that BRAF inhibition increased the expression of mitochondrial genes and mitochondrial respiration, which is associated with elevated mitochondrial oxidative stress, independent of the expression of PGC1α. Furthermore, BAY 87-2243 inhibited OXPHOS, down-regulated the expression of mitochondrial genes and increased reactive oxygen species (ROS) production. We also demonstrated that inhibition of BRAF increased the OXPHOS phenotype and therefore enhanced the effect of BAY 87-2243 on mitochondrial metabolism; the combination of both inhibitors showed synergistic effects in vitro.Interestingly, long term BRAF inhibition resulted in BRAFV600E melanoma cells to become resistant to BAY 87-2243. Preliminary studies suggest that melanoma cells pretreated with a BRAF inhibitor, with higher rates of mitochondrial respiration and elevated ROS levels, are prone to cell death when targeted by pro-oxidants. Further studies are currently ongoing to understand this resistance phenotype as well as the underlying mechanism which drives the synergistic drug effect.

Citation Format: Laura Schoeckel, Katharina Bitschar, Mélanie Héroult, Charlotte Kopitz, Andrea Hägebarth. Inhibition of mitochondrial complex I enhances the therapeutic benefit of BRAF inhibition in mutant melanoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 665. doi:10.1158/1538-7445.AM2015-665
Abstract 667: Regorafenib inhibits colorectal tumor growth through PUMA-mediated apoptosis
Authors: Chen Dongshi , Wei Liang , Yu Jian , Zhang Lin
Published: August 2015

Regorafenib, a multi-kinase inhibitor targeting the Ras/Raf/MEK/ERK pathway, has recently been approved for the treatment of metastatic colorectal cancer (CRC). However, the mechanisms of action of regorafenib in CRC cells have been unclear. We investigated how regorafenib suppresses CRC cell growth and potentiates effects of other chemotherapeutic drugs. We found that regorafenib treatment induces PUMA in CRC cells irrespective of p53 status through the NF-κB pathway following ERK inhibition and glycogen synthase kinase 3β (GSK3β) activation. Upregulation of PUMA is correlated with apoptosis induction in different CRC cell lines. PUMA is necessary for regorafenib-induced apoptosis in CRC cells. Chemosensitization by regorafenib is mediated by enhanced PUMA induction through different pathways. Furthermore, deficiency in PUMA abrogates the in vivo antitumor, antiangiogenic and chemosensitization effects of regorafenib. Our results demonstrate a key role of PUMA in mediating the anticancer effects of regorafenib in CRC cells. They suggest that PUMA induction can be used as an indicator of regorafenib sensitivity, and also provide a rationale for manipulating the apoptotic machinery to improve the therapeutic efficacy of regorafenib and other targeted drugs.

Citation Format: Dongshi Chen, Liang Wei, Jian Yu, Lin Zhang. Regorafenib inhibits colorectal tumor growth through PUMA-mediated apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 667. doi:10.1158/1538-7445.AM2015-667
Abstract 674: Early integrated stress response induction of ATF4 is required for the anticancer effects of the dual Akt/ERK inhibitor and TRAIL pathway inducer ONC201/TIC10
Authors: Kline Christina Leah , van den Heuvel Pieter , Allen Joshua E. , Dicker David T. , El-Deiry Wafik S.
Published: August 2015

ONC201/TIC10 is a first-in-class small molecule inducer of the tumor necrosis apoptosis-inducing ligand (TRAIL) pathway, inhibiting both Akt and ERK and consequently activating FOXO3a. Here we present a hitherto unrecognized and early mechanism that contributes to ONC201/TIC10's cytotoxic effects. ONC201/TIC10 activates the integrated stress response, thereby engaging the ATF4 pathway and consequently inducing expression of the pro-apoptotic proteins CHOP and DR5. Cells that are resistant to ONC201/TIC10 do not exhibit ATF4 pathway activation. Knocking down ATF4 and CHOP partially abrogates ONC201/TIC10's cytotoxicity and ONC201/TIC10-induced DR5 upregulation. We also report here that ONC201/TIC10 does not only cause apoptosis but also inhibits cell cycle progression, potentially explaining its broad-spectrum cytotoxicity against 22 out of the 23 tumor cell lines tested. ONC201/TIC10-induced cell cycle arrest was associated with downregulation of Cyclin D1 expression. ONC201/TIC10 induces CHOP and DR5 in cells regardless of whether cells are susceptible to ONC201/TIC10-induced apoptosis. Levels of the X-linked inhibitor of apoptosis (XIAP) influence whether a cell succumbs to proliferation inhibition or apoptosis in response to ONC201/TIC10. Our findings show that ONC201/TIC10 upregulates the TRAIL pathway not only by inhibiting Akt and ERK, and activating FOXO3a but also by engaging the ATF4/CHOP/DR5 pathway that leads to complementary and potent tumor cell killing. Our findings also suggest that CHOP expression may serve as a pharmacodynamic marker for ONC201/TIC10 activity.

Citation Format: Christina Leah Kline, Pieter van den Heuvel, Joshua E. Allen, David T. Dicker, Wafik S. El-Deiry. Early integrated stress response induction of ATF4 is required for the anticancer effects of the dual Akt/ERK inhibitor and TRAIL pathway inducer ONC201/TIC10. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 674. doi:10.1158/1538-7445.AM2015-674