bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 6313: A transcription block survival assay for the discovery of functional inhibitors of transcription factors
Authors: Madden Sarah K. , Mason Jody M. 
Published: September 2020

Abstract
Transcription factor dysregulation is often key to oncogenesis and cancer progression. Direct inhibition of transcription factors provides a logical therapeutic strategy but the broad and shallow interaction interfaces of their protein-protein and protein-DNA interactions are challenging to target using traditional small-molecule approaches. The larger size of peptides provides an opportunity to span such interfaces with the exquisite affinity and selectivity required, and a number of peptide-based inhibitors are starting to emerge.
In this study we focused on inhibiting basic leucine zipper transcription factors (bZIPs) as proof of principle. A key therapeutically relevant protein-protein interaction of bZIPs is the dimerization interface. Developing efficient peptide inhibitors of bZIP transcription factors has generally focused on either rational design or screen-based assays to identify peptides best able to inhibit dimerization. However, a major step in which bZIPs induce cancer initiation and progression is through DNA binding and the most effective inhibitors of bZIP dimerization do not necessarily translate into those best able to block the bZIP-DNA interaction. An assay that is able to screen for peptides that inhibit dimerization of the target transcription factor and – importantly – also selects according to the ability to inhibit protein-DNA interactions, holds significant promise in the development of more potent inhibitors.
To achieve this aim we have developed a Transcription Block Survival assay. Here key binding sites are placed into a gene essential for E.coli survival. The target transcription factor then blocks transcription of this essential gene by binding to these sites, leading to cell death. When a peptide inhibitor from a peptide library is introduced into the system that is able to effectively inhibit DNA binding of the transcription factor, the gene is transcribed, restoring growth. Libraries can have up to two million peptides and allow the assay to select for peptides that not only bind to the target transcription factor, but that are also able to differentiate peptides on their ability to inhibit the transcription factor from binding DNA.
The assay has been fully optimized for one bZIP target, and is currently undergoing the final stages of optimization for two other bZIP targets. Future work will focus on screening peptide libraries against these systems. This assay provides a route towards more efficient selection of potent inhibitors of transcription factors for the treatment of cancer.
Citation Format: Sarah K. Madden, Jody M. Mason. A transcription block survival assay for the discovery of functional inhibitors of transcription factors [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 6313.
Abstract 6316: High throughput small molecule screening with synthetic 3D lung-mimetic hydrogels in the rare lung cancer lymphangioleiomyomatosis
Authors: Pietrobon Adam , Yockell-Lelievre Julien , Doré Carole , Tam Roger Y. , Delaney Sean P. , Shoichet Molly , Stanford William L. 
Published: September 2020

Abstract
Lymphangioleiomyomatosis (LAM) is a rare lung cancer characterized by immature smooth muscle-like cell invasion of the lung parenchyma, leading to cystonodular destruction and respiratory decline. Interestingly, LAM is a monogetic disease caused by loss of TSC2, conferring constitutive mTORC1 activation. Currently, there are no approved therapeutics which exhibit LAM cell-specific cytotoxicity. Here, we describe a novel 3D drug screening platform which couples a rationally designed, synthetic, lung-mimetic hydrogel to high content imaging. We have designed and synthesized a hyaluronic acid-based hydrogel which mimics endogenous lung tissue. Human LAM cell models maintain mTORC1 activation in the hydrogel compared to matched controls, reflective of human LAM lesions. We observe LAM cells to exhibit both protease-dependent and independent modes of invasion: the former imparted by gel crosslinking with an MMP-cleavable peptide, the latter enabled by methylcellulose conjugation. Proliferation is inhibited as LAM cells actively invade through the gel. We coupled our culture system to high content confocal microscopy, permitting measurements of cell viability and invasion depth in response to therapeutic screening. We subsequently tested an 800-drug library of Health Canada-approved small molecule cancer therapeutics on LAM cells and matched controls. Surprisingly, LAM cells exhibited pan-therapeutic resistance as measured by both invasion and viability metrics. Enrichment analysis revealed categories of therapeutics which demonstrated LAM-selective cytotoxicity and/or anti-invasiveness. We performed a refinement screen on select therapeutics and identified AURA inhibition as an effective LAM cell-specific therapeutic avenue. Work is ongoing to establish the most efficacious small molecule inhibitor. In conclusion, we describe a novel drug screening platform which enables high content measurements of viability and invasion in response to drug screening in a lung-mimetic system. We have identified AURA inhibition as a potential therapeutic avenue for LAM patients using this screening platform. Future work will involve target validation, elucidation of the mechanism of action, and testing in a pre-clinical mouse model. As these small molecules are Health Canada-approved, we anticipate rapid clinical translation of our preferred candidate.
Citation Format: Adam Pietrobon, Julien Yockell-Lelievre, Carole Doré, Roger Y. Tam, Sean P. Delaney, Molly Shoichet, William L. Stanford. High throughput small molecule screening with synthetic 3D lung-mimetic hydrogels in the rare lung cancer lymphangioleiomyomatosis [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 6316.
Abstract 6331: Cloaking VEGF in small extracellular vesicles as a cause of adaptive resistance to anti-VEGF therapy
Authors: Ma Shaolin , Mangala Lingegowda S. , Hu Wen , Bayaktar Emine , Hu Wei , Jennings Nicholas B. , Coleman Robert L. , Lopez-Berestein Gabriel , Sood Anil K. 
Published: September 2020

Abstract
Despite the wide use of anti-vascular endothelial growth factor (VEGF) therapy (AVT) for many solid cancers, most of them become resistant to such therapy, resulting in progressive disease. Therefore, new biomarkers and strategies for blocking adaptive resistance of cancer to AVT are needed. Small extracellular vesicles (small EVs, with a size of &lt;200 nm) have been extensively studied as crucial intercellular messengers by delivering DNA, RNA, and proteins to recipient cells. We hypothesized that the packaging of angiogenic factors, especially VEGF, into small EVs could cause adaptive resistance to AVT by evading recognition by therapeutic antiboides. In this study, we carried out a series of in vitro (immunoblot analysis, flow cytometry, tube formation, and immunofluorescence staining) and in vivo (subcutaneous mouse models and Matrigel plug angiogenesis assay) experiments to determine the roles of small EVs in AVT resistance. We found that cancer-derived small EVs increasingly package VEGF and other factors in response to AVT. Further studies showed that bevacizumab cannot recognize the VEGF packaged inside small EVs (eVEGF) and eVEGF was protected from being neutralized by bevacizumab. Notably, eVEGF was transferred to endothelial cells, triggered intracrine signaling, and subsequently increased the number of tubes formed in an in vitro angiogenesis assay. In addition, VEGF was present at higher levels in serum small EVs in mouse models of adaptive resistance of ovarian cancer to bevacizumab than in models of sensitivity to it. Ovarian cancer cell-derived eVEGF increased tumor growth despite treatment with bevacizumab. Collectively, our data provide a new mechanism whereby eVEGF evades recognition by therapeutic antibodies and promotes tumor angiogenesis and progression. These findings have implications for biomarkers and new therapeutic strategies for ovarian cancer therapy.
Citation Format: Shaolin Ma, Lingegowda S. Mangala, Wen Hu, Emine Bayaktar, Wei Hu, Nicholas B. Jennings, Robert L. Coleman, Gabriel Lopez-Berestein, Anil K. Sood. Cloaking VEGF in small extracellular vesicles as a cause of adaptive resistance to anti-VEGF therapy [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 6331.
Abstract 6338: High-throughput screening program to target ovarian cancer leader cells
Authors: Karimnia Nazanin , Bilandzic Maree , Plebanski Magdalena , Stephens Andrew N. 
Published: September 2020

Abstract
Despite its relatively low incidence ovarian cancer is one of the most lethal gynecological malignancies, characterized by high recurrence rate following primary therapy and almost universal acquisition of chemoresistance. Recently, we identified a subpopulation of “Leader Cells” (LCs) that control invasion, progression and acquired chemoresistance. Therapies targeting leader cells are likely to be pivotal to improving prognosis, particularly in patients who are refractory to standard-of-care. To investigate the role of LCs in chemoresistance, we used CRISPR to integrate an in-frame T2A-GFP expression cassette downstream of the native LC marker gene, Keratin 14 (KRT14). Genomic integration drives the LC specific expression of GFP, facilitating visualization and quantitative tracking of the LC subset. The status of GFP as a surrogate marker of LCs in vitro was confirmed by immunostaining and expression analyses. Chemotherapeutic intervention studies and co-staining with the apoptosis marker cleaved caspase 3, demonstrated a significant enrichment of LCs that represent a non-apoptotic subset of cells resistant to standard chemotherapy. High-throughput drug screening, combining large functional drug libraries with high-throughput viability and invasion assays, was used to identify drugs that could inhibit LC function in vitro. Ovarian cancer cell lines (SKOV3 and COV362.4 expressing KRT14-T2A-GFP) were screened against 1450 FDA-approved drugs, and 218 hits identified that inhibited LC viability, invasive capacity or both. Amongst the hits that we identified, the neural signaling pathway emerged as a potential novel mediator for LC function. Further validation experiments confirmed that inhibition of the neural signaling pathway did not affect viability but synergized with chemotherapy to reduce cisplatin IC50 by 10-20-fold. Our data demonstrate that the high-throughput screen developed is a viable approach to identify opportunities for drug repurposing. Moreover, the neural signaling pathway appears to be important in LC function and inhibiting this pathway results in increased sensitivity to chemotherapy in vitro. Ongoing work will evaluate the applicability of this new tool for clinical screening programs, and further, validate existing drugs for potential repurposing.
Citation Format: Nazanin Karimnia, Maree Bilandzic, Magdalena Plebanski, Andrew N. Stephens. High-throughput screening program to target ovarian cancer leader 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 6338.
Abstract 6360: Development of a new substituted 1,3-dioxoisoindoline series that are RPS6K1 protein kinase inhibitors as potential cancer therapeutics
Authors: Sridhar Jayalakshmi , Komati Rajesh , Bratton Melyssa 
Published: September 2020

Abstract
The 40S ribosomal S6 kinase 1 (S6K1), a conserved serine/threonine protein kinase, is the principal kinase effector downstream of the PI3K/mTOR regulatory signaling pathway. Over-expression of RPS6K1 has been associated with cell transformation and elevated proliferation rates in tumors, poor prognosis and an increased risk of local recurrence. RPS6K1 has been found to play an important role in the progression of ER-positive breast cancer, HER2 positive breast cancer and node-negative premenopausal breast cancer. Overexpression of RPS6K1 is closely correlated with aggressive progression and poor prognosis in prostate cancer patients. Inhibition of this kinase can prove to be beneficial for the treatment of several types of cancer including breast cancer, prostate cancer, non-small cell lung cancer, etc. Our laboratory has identified 7-amino-5-amido-1,3-dioxoisoindolines as S6K1 inhibitors with low micromolar IC50 values (2-5 μM). These compounds also inhibited the growth of prostate cancer cells DU-145 and breast cancer cells MCF-7 (ER-positive) and SKBR3 (HER2-positive). The IC50 values of growth inhibition of SKBR3 breast cancer cell lines by the compounds RJ19, RJ22 and RJ28 are 3.3, 1.9 and 4.7 μM, respectively. The core structure 5,7-diamino-1,3-dioxoisoindoline was found to selectively inhibit RPS6K1 over 99 other oncologic protein kinases. This series of compounds have the potential to become viable therapeutics for many types of cancer. The results from the synthesis of 7-amino-5-amido-1,3-dioxoisoindoline derivatives, the structure activity studies, and their efficacy studies will be presented.
Citation Format: Jayalakshmi Sridhar, Rajesh Komati, Melyssa Bratton. Development of a new substituted 1,3-dioxoisoindoline series that are RPS6K1 protein kinase inhibitors as potential cancer therapeutics [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 6360.
Abstract 6363: Small molecule PG3-Oc partially restores the p53 pathway transcriptome and proteome in tumors with mutant p53 through ATF4 transcriptional modulation by ERK1/2 and CDK9
Authors: Tian Xiaobing , Ahsan Nagib , El-Deiry Wafik S. 
Published: September 2020

Abstract
The tumor-suppressor p53 prevents cancer development via initiating cell-cycle arrest, cell death, repair, or anti-angiogenesis processes. Over 50% of human cancers harbor cancer-causing mutations in p53. p53 mutations not only abrogate its tumor-suppressor function, but also endow mutant p53 with a gain of function (GOF), creating a proto-oncogene that contributes to tumorigenesis, tumor progression, and chemo- or radiotherapy resistance. Thus, targeting mutant p53 and/or restoring a wild-type p53 signaling pathway provides an attractive strategy for cancer therapy. We previously reported that prodigiosin, a natural product, has potent anti-cancer activity against tumors with mutated p53. The novel synthetic analogue of prodigiosin, PG3-Oc, is potent in reducing both short-term and long-term proliferation in a broad panel of mutant p53 cell lines. PG3-Oc treatment induces genome-wide restoration of p53 target genes, which are mediated by activation of ATF4. We demonstrate that ERK1/2 and CDK9 kinase functions are required for ATF4 transcriptional activity in mutant p53-expressing cancer cells. We have characterized a mechanism of PG3-Oc that engages the apoptosis pathway by upregulation of the pro-apoptotic p53 target gene PUMA in a time-and dose-dependent manner through the ATF4/PUMA axis in colorectal cancer cell lines. We further report the impact of PG3-Oc on the p53 transcriptome and proteome and the role of ATF4. Our results demonstrate feasibility of functional cellular reprogramming to restore p53 pathway signaling and provide novel insights into mechanisms of signaling leading to cell death and tumor suppression with potential clinical relevance in cancer therapy.
Citation Format: Xiaobing Tian, Nagib Ahsan, Wafik S. El-Deiry. Small molecule PG3-Oc partially restores the p53 pathway transcriptome and proteome in tumors with mutant p53 through ATF4 transcriptional modulation by ERK1/2 and CDK9 [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 6363.
Abstract 6371: Gaseous nitric oxide at high concentration is a powerful anti-tumor agent both in-vitro and in-vivo
Authors: Confino Hila , Lisi Steve , Kalaora Rinat , Goldshtein Matan , Avniel Amir 
Published: September 2020

Abstract
Nitric Oxide (NO) acts as a signaling molecule in the body in different pathologic conditions, including cancer. Beyond Air is developing an innovative gaseous Nitric Oxide (gNO) based tumor ablation method. The highly diffusible nature of NO may ease its spreading inside the tumor, while its short half-life may limit its effect to the tumor site and not affect the surrounding area.
According to our preliminary data, exposure of gNO at 10,000-50,000ppm for a very short time (in some cases one minute or less) efficiently eradicates breast, colon, lung and prostate cancers and shows a potent mouse tumor's destruction in-vivo.
Methods: Mouse colon and breast cancer cell lines were exposed to gNO at 150-50,000 ppm for 10 seconds - 180 seconds. Cell viability was examined 24hrs later by XTT assay.
Mouse colon tumors were treated by gNO at 10,000ppm - 50,000ppm intratumorally by hypodermic needles or local exposure of the tumor using a plastic box or tube.
Results: To test if gNO at 150-50,000ppm can decrease cancer cell viability, cells were exposed (I) directly to NO in a 96 well plate or (II) in an acrylic glass box. Cell viability was tested 24hrs later by XTT. According to our findings, less than 3% of 4T1 cells remain viable when exposed to 10,000ppm NO for 3 minutes or 50,000ppm NO for 1 minute in a 96-well plate. ~1% of CT26 cells remain viable after exposure to 50,000ppm NO for 10 seconds in a 96 well plate; while less than 10% remain viable after 1 minute of exposure of 50,000ppm NO in a box.
To test if gNO at 10,000-50,000ppm can destroy mouse colon cancer tumors in-vivo, tumors were exposed to gNO locally by hypodermic needles or a plastic cover. Air treated tumor bearing mice served as the control group.
A complete tumor destruction was observed when the skin was removed from the tumor and the entire tumor was exposed to 10,000ppm gNO for 10 minutes.
Conclusion: In the current work, gNO at high concentration showed a substantial cytotoxic effect on cancer cells in-vitro and in-vivo. The proposed project represents development of a novel treatment for cancer. Our innovative gNO based treatment may serve to treat solid tumors locally or be used as an adjunctive therapy for surgery and can be synergized with common anticancer therapies, such as chemotherapy and radiotherapy, to further enhance the effect.
Citation Format: Hila Confino, Steve Lisi, Rinat Kalaora, Matan Goldshtein, Amir Avniel. Gaseous nitric oxide at high concentration is a powerful anti-tumor agent both in-vitro and in-vivo [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 6371.
Abstract 6409: Targeted degradation of endogenously tagged proteins for phenotypic studies using HaloPROTAC3 and HaloTag technologies
Authors: Caine Elizabeth A. , Mahan Sarah , Riching Kristin , Murphy Nancy , McDougall Mark , Corona Cesear , Heid Chris , Daniels Danette L. , Urh Marjeta 
Published: September 2020

Abstract
To understand a protein's function inside the cell, studies are often done to remove it using CRISPR-mediated knockout or RNAi knockdown methods. These approaches have challenges in either obtaining efficient or complete loss, are not possible if the protein target is essential for cell growth, and are genetic methods, not directly protein loss. To overcome these hurdles, we have employed a highly precise and temporally controlled target protein degradation strategy utilizing HaloPROTAC, a HaloTag Proteolysis-targeting chimera small molecule which will specifically degrade HaloTag fusion proteins in live cells. HaloPROTAC3, developed by Prof. Craig Crews at Yale University, recruits HaloTag fusion proteins to VHL E3 ligase complexes, resulting in ubiquitination and degradation of the HaloTag fusion via the ubiquitin-proteasomal pathway. For phenotypic studies, removal of the endogenous proteins is often required, therefore we have developed efficient protocols for the introduction of HaloTag via CRISPR/Cas9 gene editing into either the endogenous N- or C-terminal loci of any target protein. We have additionally appended the 11 amino acid HiBiT to the HaloTag-target protein, allowing for highly quantitative and kinetic monitoring of degradation to be tracked in live cells without the use of antibodies. Shown here are several key therapeutic proteins, BRD4 and B-catenin, which have been endogenously tagged with HiBiT-HaloTag via CRISPR/Cas9 and show rapid and sustained degradation (80-90% loss) after treatment with HaloPROTAC3. For BRD4, this loss was similar to what was observed using a BET-family specific PROTAC. For B-catenin, further phenotypic studies were done to show a muted response to Wnt3a stimulation and TCF/LEF promoter gene activation after degradation of endogenous B-catenin. These examples demonstrate that the technologies can be used readily to elicit robust degradation of target proteins, with control over the protein level and the time frame for which the protein will be lost, in order to more fully understand protein function. Together, these provide new opportunities for phenotypic studies based directly on control of protein levels and loss, with the ability to study the roles of essential proteins not amenable to genetic knockout or knockdown approaches.
Citation Format: Elizabeth A. Caine, Sarah Mahan, Kristin Riching, Nancy Murphy, Mark McDougall, Cesear Corona, Chris Heid, Danette L. Daniels, Marjeta Urh. Targeted degradation of endogenously tagged proteins for phenotypic studies using HaloPROTAC3 and HaloTag technologies [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 6409.