bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 6293: Novel approach for automated sequential immunoassay for quantitation and characterization of PI3K-Akt pathway proteins
Authors: Kazakova Irina G. , Tran Crystal , Silver Anita , Dermody Jessica 
Published: September 2020

Abstract
The PI3K-Akt signaling pathway modulates cell growth, survival and apoptosis, and this pathway is frequently altered in human cancers, contributing resistance to radiation and chemotherapy treatment. Akt is a target for specific inhibition and recently, a number of small molecules have been developed to improve the pharmacologic properties of known inhibitors like wortmannin and LY294002. However, pan-Akt inhibitors can result in unanticipated side effects due to the lack of specificity for Akt isoforms 1, 2, and 3. Therefore, detection and quantitation of Akt isoforms and their downstream targets for both expression levels and phosphorylation states is crucial for therapeutic drug development. Here we demonstrate application of the Multiple Detection Approach to characterize the PI3K-Akt signaling pathway. This approach uses sequential analysis of proteins separated and immobilized in a capillary, by performing either dual immunoassays or immunoassay with total protein on the Simple Western platform using chemiluminescence detection. Assays with control and LY294002 inhibitor-treated samples were developed. Proteins were first separated based on molecular weight via capillary electrophoresis, followed by immobilization via UV-crosslinking. Next, PI3K-Akt pathway targets were sequentially probed in the same capillary with total and phospho-specific antibodies, to determine the phosphorylated fraction relative to the total fraction. Primary antibodies from the first immunoprobe were removed after the detection step with &gt;95% efficiency, as confirmed by re-probing with the same secondary antibody. Target protein loss was negligible due to covalent immobilization to the capillary wall, which was confirmed with re-probing, thus validating the quantitative data generated using this sequential approach. In addition, total protein normalization was performed in tandem with the immunoassay in the same capillary. This approach enables normalization of phosphorylation levels and/or target abundance in cell line or tissue samples, correcting for change in protein content due to treatment, loading, and/or other systematic errors. These results present the utility of the Multiple Detection Approach to quickly characterize and quantify proteins involved in signaling pathways targeted during development of cancer therapies.
Citation Format: Irina G. Kazakova, Crystal Tran, Anita Silver, Jessica Dermody. Novel approach for automated sequential immunoassay for quantitation and characterization of PI3K-Akt pathway proteins [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6293.
Abstract 6294: Absolute, quantitative and high throughput measurement of tissue biomarkers in FFPE specimens using QDB method
Authors: Zhang Jiandi 
Published: September 2020

Abstract
Absolute quantitation of tissue biomarkers in FFPE samples to aid drug discovery efforts. Tissue protein biomarkers play irreplaceable role in clinical diagnosis, prognosis and prediction. It is also heavily used in drug discovery to evaluate the effectiveness of a drug candidate, or to monitor its potential side effects at both preclinical stage and during clinical trials. In recent years, several tissue protein biomarkers are used as companion diagnostics to identify right patients for the right treatment.
However, while the consistency and objectivity are the prerequisites for any assays to be used in scientific studies, the current prevailing method for tissue biomarker assessment, immunohistochemistry (IHC), is known to be associated with inconsistency and subjectivity. For example, there were studies demonstrating as much as 20% inaccuracy in Her2 assessment for breast cancer patients using IHC method. Conceivably, this practice leads to significantly increased cost and efforts in drug discovery.
In this study, we demonstrated the usage of Quantitative Dot Blot (QDB) method to achieve absolute and quantitative measurements of tissue biomarkers in Formalin Fixed Paraffin Embedded (FFPE) samples in high throughput format. We were able to develop multiple QDB-based immunoassays using clinically validated antibodies for IHC in several types of cancers including breast cancer, lung cancer and prostate cancer. In the case of Her2, when the quantitatively measured results were converted into dichotomous variables using Receiver Operative Characteristics, we were able to achieve concordance with IHC at 94.8% (n=748), and 94.2% with Fluorescence in situ hybridization (FISH) (n=141). QDB-based assays also require minimum amount of total protein lysates, ranging from 0.1 to 0.5 μg per sample. Total protein lysates extracted from 2X5μm is sufficient to measure at least 5 to 10 protein biomarkers in triplicate.
The introduction of QDB method in drug discovery promises significantly increased objectivity and consistency of the results. The high throughput aspect of QDB method also suits well the daily activity in drug development. We believe the adoption of this method should significantly speed up the drug discovery process while reduce the related costs and efforts.
Citation Format: Jiandi Zhang. Absolute, quantitative and high throughput measurement of tissue biomarkers in FFPE specimens using QDB method [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6294.
Abstract 6310: Ex vivo phosphoflow cytometry to capture fast changes of phospho-proteins at single cell level
Authors: Zhang Qiyao , Wang Jingjing , Ge Yun , Xiong Yajie , Qin Beibei , Gu Qingyang , Ji Qunsheng 
Published: September 2020

Abstract
BACKGROUND: Protein phosphorylation is an important cellular regulatory process and a key indicator of signaling activation or deactivation. However, the fast switch-on/off of the process makes difficult the timely and accurate detection of the phosphorylation status. It is even more challenging to monitor the protein phosphorylation during cancer immune response in vivo, whereas immune responses vary among different cell types and can sometimes only be detected in certain rare populations like Treg (T regulatory) and TCM (T central memory) cells. The ability of Phosphoflow cytometry to accurately and rapidly detect many phospho-protein targets simultaneously at single cell level provides significant advantages over conventional methods. Ex vivo phosphoflow cytometry is increasingly being used as a tool for the discovery of biomarkers used in monitoring of disease and therapy progress.
RESULTS: We established an ex vivo phosphoflow system to evaluate fast changes of pStat5 at single cell level in mouse models. Method validation was carried out step by step to ensure the reproducibility of this assay. The method development included optimizing gating strategy for non-phosphorylated proteins, selecting the reagents and optimizing the procedures of Lyse/Fix, perm, setting up positive controls under in vitro and ex vivo conditions, and optimizing phosphorylated protein staining and detection. Our data showed that the test articles induced differential pStat5 expression in different cell types, and in a dose-dependent manner.
SUMMARY: We have established reliable ex vivo phosphoflow method to detect the fast changes of phospho-proteins at single cell level in animal models. This powerful method will enable us to examine protein post-translational modification in complicated host systems.
Citation Format: Qiyao Zhang, Jingjing Wang, Yun Ge, Yajie Xiong, Beibei Qin, Qingyang Gu, Qunsheng Ji. Ex vivo phosphoflow cytometry to capture fast changes of phospho-proteins at single cell level [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6310.
Abstract 6323: Identification of genomic alterations related to treatment progression in RWD
Authors: Chaudhary Nayan , Luhn Patricia , Jansen Gunther , Metcalfe Ciara , Hafner Marc 
Published: September 2020

Abstract
Real-world data (RWD) are potentially a highly valuable resource to accelerate drug discovery and improve clinical practice. In particular, datasets combining clinical outcomes and comprehensive genomic profiling (CGP) could uncover new mechanisms of resistance and identify new patient populations for targeted therapies. Here, we used a de-identified clinico-genomic database (CGDB) from routine clinical care based on a clinical EHR-derived data from Flatiron Health linked to tumor sequencing results from Foundation Medicine to assess how prevalence of genomic alterations changes following progression on targeted therapies in the metastatic setting.
One of the main challenges we faced is the lack of systematic and longitudinal genomic testing: among the patients receiving a specific treatment, some had a CGP performed prior to treatment, others only around the end of the line of therapy, but only a handful had multiple tests. We therefore established a methodology to assess changes in alteration prevalence and tested how our results are sensitive to cohort building parameters such as timing of testing, duration of therapy, or prior lines of treatment. Our approach successfully recovered three established cases of therapeutic resistance: (1) EGFR T790M in non-small cell lung cancer patients treated with erlotinib or gefitinib; (2) AR alterations following hormonal therapies in prostate cancers; and (3) ESR1 mutations in hormone-receptor positive breast cancer patients progressing on endocrine therapies other than fulvestrant. We delved into the latter case and identified differences in the type of ESR1 mutation that occurred following endocrine therapies if CDK4/6 inhibitors were co-administered or not.
On one hand, RWD lack randomization and can thus be subject to confounding variables but on the other hand, RWD have the advantage of including a larger number of patients in a real-world setting compared to clinical trials. Our analysis shows that real-world CGDB can recapitulate the expected emergence of mutations following specific lines of treatment. Our study is a proof of concept of usage of RWD to assess how prevalence of genomic alterations changes following treatment. Our method allowed us to assess differences between patient cohorts and generate novel hypotheses on treatment-related changes in genomic profiles, which can ultimately lead to new drug indications.
Citation Format: Nayan Chaudhary, Patricia Luhn, Gunther Jansen, Ciara Metcalfe, Marc Hafner. Identification of genomic alterations related to treatment progression in RWD [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6323.
Abstract 6327: Artesunate reduces tumor growth and induces different kinds of cell death in docetaxel-resistant prostate carcinoma cells
Authors: Braeunig Vitus , Schupp Patricia , Markowitsch Sascha , Vakhrusheva Olesya , Erb Holger , Efferth Thomas , Haferkamp Axel , Juengel Eva 
Published: September 2020

Abstract
Introduction: Prostate carcinoma (PCa) is the most common cancer in men. The therapeutic effect of approved compounds, such as docetaxel, is limited due to the development of therapy resistance, making new treatment options essential. Artesunate (ART), used in Traditional Chinese Medicine, has shown anti-tumor activity in several tumor types. However, little is known about the efficacy of ART on therapy-resistant PCa. Therefore, the impact of ART on docetaxel-resistant PCa cells was investigated.
Material and Methods: Parental (=sensitive) and docetaxel-resistant PCa cell lines, PC3, DU145, and LNCaP, were exposed to ART [1-100 µM] for 24, 48, or 72 hours. Cells not exposed to ART served as controls. Tumor cell growth, proliferation, cell cycling, and the expression of cell cycle regulating proteins were assessed. Apoptotic, ferroptotic, and necrotic effects were also evaluated.
Results: Exposure to ART significantly reduced tumor cell growth and proliferation of parental and docetaxel-resistant PCa cells in a time- and dose-dependent manner. Cell cycle arrest in the G0/G1 phase after 48h exposure to 37.5 µM ART was observed in parental LNCaP and in all three resistant cell lines. The observed changes in cell growth and cell cycle phases were accompanied by marked modulation of cell cycle regulating proteins. Significant apoptotic effects were observed in parental PC3 and both parental and resistant LNCaP, whereas ferroptosis was apparent in parental and resistant DU145 after exposure to ART. Necrotic events were not detectable in the PCa cells after ART exposure.
Conclusion: ART induced anti-tumor effects, though differing according to the cell line, were apparent in not only parental but also in docetaxel-resistant PCa cells. Thus, adding ART to standard therapies might be a promising treatment strategy for patients with advanced prostate cancer. Further investigations are necessary to verify our findings.
Citation Format: Vitus Braeunig, Patricia Schupp, Sascha Markowitsch, Olesya Vakhrusheva, Holger Erb, Thomas Efferth, Axel Haferkamp, Eva Juengel. Artesunate reduces tumor growth and induces different kinds of cell death in docetaxel-resistant prostate carcinoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6327.
Abstract 6349: A subgroup of potent CB002 xanthine derivatives define a novel class of anti-cancer drugs that restore the p53 pathway in tumors with mutated p53 and target an S-phase checkpoint
Authors: Borrero Liz Janice Hernandez , Dicker David T. , El-Deiry Wafik S. 
Published: September 2020

Abstract
Tumor suppressor TP53 is a transcription factor found to be mutated in over 50% of human tumors. Unique to other tumor suppressors, mutations in TP53 result in a stable protein that acquires a gain-of-function activity leading to an aggressive tumor with more metastatic potential and resistance to chemotherapy. The TP53 gene codes for the p53 protein which controls cell survival fate depending on the type of cellular stress signals. A well-known DNA damage stress response signaling pathway involves the activation of ATR/ATM and phosphorylation of Chk1/Chk2 leading to p53 activation and cell cycle arrest. As of now, there are no FDA approved drugs that restore the p53-pathway in any tumors. We previously discovered the parental compound CB002 along with its analogs that are capable of restoring the p53-pathway in tumors with mutated p53. These compounds are xanthine analogs but unlike known xanthines such as caffeine, pentoxifylline and theophylline, they restore the p53 pathway, including upregulation of Noxa, but do not deregulate the G2 checkpoint. Rather, 24 hr treatment in DLD-1 and SW480 cells, shows that our analogs induce p53 transcriptional targets whereas caffeine, pentoxifylline and theophylline do not. In addition, our cell cycle analysis data in synchronized SW480 cells treated with etoposide suggests that CB002 and its analogs perturb the S-phase of the cell cycle and do not deregulate the G2 checkpoint. This is also evidenced by increased p-RPA/RPA2 and cyclin E, and decreased p-histone 3 protein expression. Our microarray data indicates that our p53-pathway restoring xanthine analogs downregulate essential proteins of the DNA synthesis and repair machinery. We are currently investigating the involvement of key kinases and transcriptional factors regulated in S-phase following treatment with CB002 and its more potent analogs. Our data also suggests the activation of ATR in response to CB002 analog treatment in SW480 cells at 24 hrs. We are evaluating the benefit of synthetic lethality of these analogs in BRCA-deficient cell lines, focusing on target identification, determining the proteomic changes and in vivo efficacy of our p53-pathway restoring CB002 xanthine analogs. Taken together, our data suggests that CB002 and its potent xanthine analogs may provide a unique therapeutic strategy that can be clinically translated.
Citation Format: Liz Janice Hernandez Borrero, David T. Dicker, Wafik S. El-Deiry. A subgroup of potent CB002 xanthine derivatives define a novel class of anti-cancer drugs that restore the p53 pathway in tumors with mutated p53 and target an S-phase checkpoint [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6349.
Abstract 6351: A novel NSC small molecule inhibitor, inhibits proliferation and invasion of triple-negative breast cancer cells
Authors: Yousefi Hassan 
Published: September 2020

Abstract
Treatment of patients with Triple-negative breast cancer (TNBC) has been challenging due to the absence of well-defined molecular targets and high invasive and proliferative capacities of these cells. Current treatments against TNBC have shown minimal activity due to the high recurrence rate in the patients. There is, therefore, a pressing need for novel and efficacious therapies against TNBC. Here, we found a novel small molecule inhibitor with potent anti-tumor activity against TNBC cells. Anti-proliferative effects of NSC small molecule inhibitor were determined using 2D and 3D culture cell proliferation. Propium iodide staining was performed to analyze cell cycle distribution and Annexin/PI assay was carried out to evaluate the induction of apoptosis. Western blot analyses, proteomic and gene array techniques were applied to elucidate the molecular mechanisms underlying the anti-tumor activity of the small molecule inhibitor. Our data show that our NSC small molecule inhibitor, reduced the proliferation of a panel of breast cancer cells, particularly, TNBC cells through the induction of apoptosis but not cell cycle arrest. Proteomic and gene array analysis implicated a myriad of canonical signaling pathways including cell death, and cell survival. Furthermore, we found that treatment with NSC small molecule inhibitor impaired TNBC cell migration and invasion via expression reduction of the epithelial-to-mesenchymal transition (EMT) markers. In addition, we found that NSC small molecule inhibitor decreased TNBC cell proliferation and clonogenic survival and induced apoptotic cell death through inhibition of the anti-apoptotic proteins. Altogether, our data indicate that NSC small molecule inhibitor may exhibit anti-tumor activity in TNBC cells and provide a rationale for further investigation of the potential of NSC small molecule inhibitor as an attractive therapeutic drug for TNBC. We are currently in the process of validating our proteomic data. Further in-depth investigations are required to evaluate the exact targets and mechanism of this potent small molecule inhibitor, and its anti-neoplasm effects on TNBC in vivo.
Citation Format: Hassan Yousefi. A novel NSC small molecule inhibitor, inhibits proliferation and invasion of triple-negative breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6351.
Abstract 6361: Identification of mitochondrial RNA polymerase inhibitors via deep convolutional neural networks: A therapeutic strategy for cancer metabolism
Authors: Khalid Noor-Ul-Ain , Warrington Jeffrey M. , Laposa Rebecca Rachelle 
Published: September 2020

Abstract
Dysregulated metabolism is a hallmark of cancer and a substantial subset of many cancers rely on oxidative phosphorylation (OXPHOS) for growth and viability, including AML, breast cancer, lung cancer, glioblastoma, ovarian and pancreatic cancer. The electron transport chain (ETC) in mitochondria contains both nuclear DNA- and mitochondrial DNA-encoded subunits. Mitochondrial DNA-encoded subunits are essential for ETC function and are transcribed by the mitochondrial RNA polymerase POLRMT. We have previously validated POLRMT as an anticancer target in acute myeloid leukemia models. The challenge of inhibiting POLRMT with small molecules will arise from both a) selectivity to off targets and b) transport of the drug to the mitochondria. There exist several small nucleoside / nucleotide analogues that are known to inhibit RNA polymerase. However, this target space is rife with the opportunity for off-target activities and toxicity. Therefore, we targeted a novel pocket with the aim of finding novel chemical matter. Using AtomNet, an artificial intelligence platform that utilizes deep convolutional neural networks to predict small molecule recognition, a virtual library of 8 million compounds was screened against POLRMT. The top 72 hit compounds were tested in OCI-AML2 human acute myeloid leukemia cells for a potential decrease of mitochondrial RNA levels (MT-ND1) using reverse transcriptase polymerase chain reaction (RT-PCR). OCI-AML2 cells were treated with compounds at 0.1, 1 or 10 micromolar for 72 hours, 3 days or 6 days or with the positive control POLRMT ribonucleoside analogue inhibitor, 4'-azidocytidine. Gene expression was normalized to 18S. Twelve compounds demonstrated a time-dependent decrease in mitochondrial gene expression. In summary, these data indicate that functional inhibitors of human POLRMT can be identified rapidly via deep convolutional neural networks and machine learning techniques. Financial relationships: RL received an Artificial Intelligence Molecular Screening Award from Atomwise, and funding from the Centre for Collaborative Drug Research, Toronto, Canada. JW is an employee of Atomwise.
Citation Format: Noor-Ul-Ain Khalid, Jeffrey M. Warrington, Rebecca Rachelle Laposa. Identification of mitochondrial RNA polymerase inhibitors via deep convolutional neural networks: A therapeutic strategy for cancer metabolism [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6361.