bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2858: DEPDC1B functions as an oncogene in promoting melanoma growth and metastasis
Authors: Hu Feng , Fong Ki On , Cheung May Pui Lai , Yang Xintao , Cheung Martin
Published: July 2021

Background: Melanoma is one of the most aggressive skin cancers due to its therapeutic resistance, and high capacity to metastasize as a late manifestation. The limited effectiveness of the current treatment regimen prompts us to identify new druggable targets in governing the systemic spread of melanoma. Despite the oncogenic role of DEPDC1B in different types of cancer, its functional importance in melanoma growth and metastasis are not fully revealed.

Methods: The expression of DEPDC1B mRNA levels between skin cutaneous melanoma patients and normal samples were compared using TCGA database. Surgically procured tumor specimens from melanoma patients were obtained to investigate the protein levels of DEPDC1B expression by immunofluorescence. Melanoma cell lines WM266-4 and SK-MEL-28 were used for loss- and gain-of-function studies of DEPDC1B in AlamarBlue, EdU staining, colony formation and trans-well invasion assays. Subcutaneous and intravenous injection of melanoma cells treated with either DEPDC1B knockdown (KD) or DEPDC1B overexpression into immunodeficient mice were conducted to examine the effects on tumor growth and lung metastasis respectively.

Results: The expression levels of DEPDC1B mRNA in melanoma biopsies were higher than normal samples and were negatively corelated with both overall survival and disease-free survival. Loss-of-function studies showed that DEPDC1B KD reduced the growth rate, proliferation capacity and colony formation ability of melanoma cells. Consistently, DEPDC1B KD cells gave rise to smaller xenograft tumors and fewer number of lung nodules. In contrast, DEPDC1B overexpression promoted melanoma cell proliferation, colony formation and invasion in vitro, and enhanced xenograft tumor growth and lung metastasis.

Conclusion: The results obtained so far indicate that DEPDC1B acts as an oncogene in promoting melanoma cell growth and metastasis, suggesting a potential drug target in melanoma treatment.

Citation Format: Feng Hu, Ki On Fong, May Pui Lai Cheung, Xintao Yang, Martin Cheung. DEPDC1B functions as an oncogene in promoting melanoma growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2858.
Abstract 2868: A cell based phenotypic assay platform for cancer metastasis drug discovery and diagnostics
Authors: Chowdhury Arnab Roy , Chowdhury Debabani Roy , Pandre Manoj , Roy Samrat , Kannan Sundarajan , Ellingboe John W.
Published: July 2021

Metastatic cancers are difficult to treat and are the cause of about 90% of cancer related deaths. Furthermore, predictive diagnostics are not readily available, and lack of good translational assays has impaired drug discovery. Mestastop has developed a platform of cell based functional assays that aims to address these drug discovery and diagnostic gaps. The platform is comprised of a set of 14 cell based phenotypic assays that capture multiple aspects of the complex process of metastasis, including characterization of the plasticity ratios (EMT, MET) and stemness of cells, invasion, intravasation, circulation, extravasation, and survival in secondary tissues (e.g metabolic shift, exosome secretion). Wild type tumor cells (initially a set of colon tumor cell lines) are utilized in the platform. Non-metastatic wild type cells (e.g. HT-29) that have been engineered into metastatic cells are also utilized to further elucidate metastatic processes, thereby creating two baseline data sets for each assay; one for the normal growing tumor and the other for the select metastatic population. Patient derived tumors are now being analyzed in the platform to help build a translational model. Preclinical animal models are being jointly developed with Immunobiome Inc. (South Korea). As the data from all these assays present a multivariate problem, we are developing a machine learning algorithm, METSCANTM, to analyze the results. The platform will be applied to the screening of novel compounds for drug discovery and of approved drugs for repurposing. Analysis of patient derived tumors may allow for the early detection of patients with high metastatic potential. Four colon cancer cell lines and several standard chemotherapy drugs have been used to validate the platform. A group of triple negative breast cancer cell lines is now being tested. Preliminary data for a set of approved, marketed drugs has further validated the potential of the platform for drug discovery and repurposing. The Mestastop proprietary assay platform will empower biopharma in the discovery of novel drug candidates and repurposing of approved drugs for the treatment of metastasis, and the early diagnosis of cancers with high metastatic potential, thereby addressing the unmet need in the area of cancer metastasis.

Citation Format: Arnab Roy Chowdhury, Debabani Roy Chowdhury, Manoj Pandre, Samrat Roy, Sundarajan Kannan, John W. Ellingboe. A cell based phenotypic assay platform for cancer metastasis drug discovery and diagnostics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2868.
Abstract 2876: Adhesion GPCR CD97/ADGRE5 facilitates invasion in patient-derived glioma stem cells and induces changes in EGFR and Sox2 expression
Authors: Slepak Tatiana I. , Eichberg Daniel G. , Pascoini Ana L. , Komotar Ricardo J. , Ivan Michael E.
Published: July 2021

The main obstacles for effective treatment of Glioblastoma (GBM) are high invasiveness and heterogeneity. Each GBM tumor is composed of multiple genetically distinct subpopulations of Glioma Stem Cells (GSCs), exhibiting high cell plasticity affecting their invasive and proliferative properties. Treatment interventions and changes in microenvironment force GSCs to adapt through gene expression that supports therapeutic resistance. Expression level of the surface receptor CD97/ADRGE5 positively correlates with poor GBM patient prognosis, but its role in this tumor has not been elucidated. Here, we examined the function of CD97 in primary GSCs derived from five GBM tumors, which belong to three major genetic subtypes. We manipulated CD97 levels in these GSCs by knockdown and overexpression and analyzed the following: (i) expression of stem and subtype markers, (ii) in vitro invasive properties, (iii) cell proliferation, (iv) expression of the key proteins that regulate GSCs cellular state and influence their adaptive behavior. Our data showed that CD97 knockdown in GSCs of mesenchymal and classical subtype reduced their invasion rate in vitro. CD97 signaling positively affected expression of Epidermal Growth Factor Receptor, the main driver of astrocyte-like fate in GSCs and changed the expression of the astrocytic marker GFAP. Finally, we discovered a possible link between CD97 and expression of transcription factor Sox2, the key regulator of the endothelial-to-mesenchymal transition process that plays a crucial role in initiation of cancer cell invasion.

Citation Format: Tatiana I. Slepak, Daniel G. Eichberg, Ana L. Pascoini, Ricardo J. Komotar, Michael E. Ivan. Adhesion GPCR CD97/ADGRE5 facilitates invasion in patient-derived glioma stem cells and induces changes in EGFR and Sox2 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2876.
Abstract 288: Revisiting small molecules that influence the nuclear translocation of thioredoxin-1, prohibitin, and galectin-3 for cancer chemotherapy
Authors: Fortin Jessica S. , Wineland Thomas E. , Duggan Kelsey E. , C.-Gaudreault René
Published: July 2021

Phenyl-3-(2-chloroethyl)ureas (CEU) are alkylating agents exhibiting potent antiproliferative properties both in vitro and in vivo. CEUs' cytoxicity is not influenced by hypoxia or other clinically relevant mechanisms of chemoresistance, such as alteration of topoisomerase II activity and increased P-glycoprotein over-expression, DNA repair, intracellular gluthatione-S-transferase activity. A first subset of CEU exemplified by the prototypical molecules 4-tert-butyl-CEU (tBCEU) and 4-iodo-CEU (ICEU) have been shown to alkylate the colchicine-binding site on βII-tubulin. In parallel, these molecules disrupted the cytoskeleton and arrested the cell cycle in G2/M leading to anoikis. So far we have conducted several structure-activity relationship and molecular pharmacology studies in an attempt to refine the specificity of CEU for β-tubulin and the colchicine-binding site. Unexpectedly, a prototypical molecule designated as 1-(2-chloroethyl)-3-(4-cyclohexylphenyl)urea (cHCEU) emerged as arresting the cell cycle in G0/G1 phase instead of G2/M. Experiments using [14C-urea]-cHCEU showed its binding to thioredoxin isoform-1 (Trx-1) and prohibitin (PHB), but not β-tubulin. Complementary immunocytofluorometry experiments clearly showed the abrogation of the nuclear translocation of Trx-1 from the cytosol. We report herein that in addition to Trx-1 and PHB, cHCEU also alkylates galectin-1 and -3 isoforms. The cellular localization of thioredoxin-1, prohibitin, and galectin-3 (Gal-3) is changed following cHCEU treatment. In addition, the expression of Trx-1, PHB, and Gal-3 seems unaffected by CEU and its analogues. Interestingly, these proteins are all known to play an important role in the cell cycle regulation through various mechanisms. The mechanism(s) underlying the effect of cHCEU on protein translocation is unknow and might be relevant to design new potent anticancer drugs that will target specific and lethal biological pathways essential to tumor growth.

Citation Format: Jessica S. Fortin, Thomas E. Wineland, Kelsey E. Duggan, René C.-Gaudreault. Revisiting small molecules that influence the nuclear translocation of thioredoxin-1, prohibitin, and galectin-3 for cancer chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 288.
Abstract 2881: Snail-mediated regulation of MMP1 expression in the context of ovarian cancer invasiveness
Authors: Suzuki Tise , Curow Casey , Wang Hanmin , Martinez Asis , Hojo Nozomi , Unternaehrer Juli
Published: July 2021

Ovarian cancer ranks fifth among the causes of cancer death in US women. Despite initial positive response to cytoreductive surgery and chemotherapy treatments with platinum-based therapies, over 80% of patients with high-grade serous ovarian cancer (HGSOC), the most common type of ovarian cancer, exhibit relapse. Most recurrent cases of HGSOC are resistant to chemotherapy, and cells are highly invasive. Therefore, further studies are required to understand the mechanisms of invasiveness and chemoresistance in relapse patients. In HGSOC, invasiveness can result when epithelial cancer cells undergo epithelial-mesenchymal transition (EMT). The transcription factor SNAI1 (Snail) initiates the EMT process and increased Snail expression can result in enhancement of metastatic potential, acquisition of stem cell-like characteristics, and resistance to chemotherapeutic drugs. However, the mechanism of activation of invasiveness in ovarian cancer by Snail has not been defined. In OVCAR8, a HGSOC cell line, comparison of shSnail knockdown to control through RNA-sequencing revealed that Snail expression is directly correlated to matrix metalloprotease 1 (MMP1) expression. MMP1 is an extracellular matrix remodeler known to digest interstitial collagens. High MMP1 expression has been linked to increased cancer invasion, metastasis, angiogenesis, and apoptosis. Furthermore, in breast cancer, MMP1 has been reported to be a downstream target of Slug, another major EMT transcription factor. Some evidence for Snail activation of MMPs in liver cancer has been presented, but a direct relationship has not been established. Therefore, we hypothesize that overexpression of Snail in ovarian cancer cells results in MMP1 upregulation, leading to increased invasion. RT-qPCR was performed to analyze relative mRNA expression levels of Snail and MMP1 in eight cell lines. We found that Snail and MMP1 levels were higher in the more mesenchymal-like cell lines, some of which are known to be more invasive. Additionally, Snail overexpression led to a significant corresponding increase in MMP1 mRNA expression and resistance to cisplatin. Chromatin immunoprecipitation (ChIP) experiments demonstrate Snail binding to the MMP1 promoter. Thus, our preliminary findings have shown strong evidence of a Snail-mediated regulation of MMP1 expression in the context of HGSOC invasiveness.

Citation Format: Tise Suzuki, Casey Curow, Hanmin Wang, Asis Martinez, Nozomi Hojo, Juli Unternaehrer. Snail-mediated regulation of MMP1 expression in the context of ovarian cancer invasiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2881.
Abstract 2898: Rac1 as a driver and therapeutic target in ovarian cancer
Authors: Rivera Melanie , Grimes Martha M. , Hudson Laurie G. , Wandinger-Ness Angela
Published: July 2021

Aberrant Rac1 signaling contributes to cancer growth and metastasis, therefore, we investigated the effect of altering Rac1 expression and activity to pinpoint which metastatic processes and tumor microenvironment immune factors are influenced by Rac1 signaling. We have identified that the R- enantiomer of ketorolac, a drug given for pain management, is a Rac1 and Cdc42 selective inhibitor. In a retrospective study, we found that treatment with racemic ketorolac significantly increased the survival probability of ovarian cancer patients. RNA seq analyses of ascites tumor cell samples from ovarian cancer patients in a prospective study receiving racemic ketorolac for clinically indicated use in pain relief, showed significant downregulation of genes involved in endocytosis, regulation of actin cytoskeleton, ER protein processing, TNF and NOD signaling. To further distinguish the activities most directly regulated by Rac1 and important in ovarian cancer, we tested the impact of manipulating the expression of Rac1 in ovarian cancer cells on adhesive and invasive cell behaviors, as well as the impacts of gene expression relative to ketorolac treatment. Using electric cell-substrate impedance sensing (ECIS), Rac1 overexpression resulted in increased cell-cell barrier formation and cell adhesion. Whereas, CRISPR-CAS9 mediated knockdown of Rac1 resulted in decreased cell adhesion. Using Matrigel invasion chambers and mesothelial clearance assays, Rac1 overexpression led to increased ovarian cancer cell invasion when compared to parental and Rac1 knockdown cell lines. Furthermore, intraperitoneal growth of ovarian cancer cells overexpression Rac1 relative to parental control lines increased tumor burden in vivo. Conversely, with knockdown of Rac1, tumor burden and omental adhesion was decreased, as quantified using IVIS bioluminescence imaging. RNA seq analyses of tumors manipulated for Rac1 as compared to R-ketorolac will be presented. The composite data indicate that targeting Rac1 in ovarian cancer with R-ketorolac or otherwise has therapeutic benefit in decreasing tumor cell dissemination and metastasis.

Citation Format: Melanie Rivera, Martha M. Grimes, Laurie G. Hudson, Angela Wandinger-Ness. Rac1 as a driver and therapeutic target in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2898.
Abstract 2901: Pdcd4 inhibits slug translation to suppress E-cadherin expression and invasion in colon tumor cells
Authors: Wang Qing , Zhu Jiang , Yang Hsin-Sheng
Published: July 2021

Programmed cell death 4 (Pdcd4) has been demonstrated to function as a tumor suppressor and protein translation inhibitor. We previously reported that knockdown of Pdcd4 down-regulated E-cadherin expression. E-cadherin is a key regulator for tumor cell invasion and metastasis, whose expression is controlled by transcription repressors Snail, Slug, ZEB1/2, and Twist. Both Slug and Snail have been shown to repress E-cadherin expression and contribute to tumor progression in various cancers including colon cancer. Here, we reported that knockdown of Pdcd4 increased Slug protein level but not SLUG mRNA level in colon tumor cells. Conversely, over-expression of Pdcd4 decreased Slug expression. Using sucrose gradient fractionation assays, Pdcd4 knockdown was found to increase the population of SLUG mRNA in the polysomal fractions. These results indicate that Pdcd4 translationally regulates Slug expression. To understand the mechanism by which Pdcd4 regulates Slug translation, the SLUG 5' untranslated region (5'UTR) was cloned and fused with luciferase reporter and named as 5'UTR-Slug-Luc. Knockdown of Pdcd4 significantly enhanced the translation of 5'UTR-Slug-Luc while ectopic expression of Pdcd4 repressed the translation of 5'UTR-Slug-Luc, suggesting that SLUG 5'UTR is an essential element for Pdcd4 to inhibit Slug translation. Moreover, cells treated with silvestrol, an inhibitor for translation initiation factor 4A (eIF4A), significantly suppressed Slug translation. In addition, ectopic expression of Pdcd4 inhibited Slug translation and thereby decreased E-cadherin expression. By contrast, Pdcd4(D418A), a mutant that does not bind with eIF4A, failed to suppress the expression of Slug and E-cadherin. These findings suggest that Pdcd4-inhibited Slug translation is through suppressing eIF4A. Furthermore, knockdown of Slug in Pdcd4 knockdown cells restored the E-cadherin expression and reversed the invasive capacity induced by Pdcd4 knockdown, indicating that up-regulation of Slug contributes to Pdcd4-knockdown enhanced invasion. Taken together, this study demonstrates that Pdcd4 inhibits translation of Slug to regulate E-cadherin expression and invasion in colon tumor cells.

Citation Format: Qing Wang, Jiang Zhu, Hsin-Sheng Yang. Pdcd4 inhibits slug translation to suppress E-cadherin expression and invasion in colon tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2901.
Abstract 2905: The relationship between peritoneal dissemination of gastric cancer & exosome
Authors: Kinoshita Kazuya
Published: July 2021

Introduction: In recent years, studies of exosomes have progressed rapidly, suggesting a connection to various physiological functions and pathogenesis. The exosomes are small extracellular vesicles with a diameter of 30 to 100 nm surrounded by lipid bilayers, and are attracting attention as a new cell-cell communication medium for carrying lipids, proteins, RNA, etc. in vivo. In addition, in exosomes released by cancer cells, it contains a large number of molecules related to angiogenesis and immunosuppression. It is believed to contribute to building microenvironments suitable for cancer cell growth and promoting cancer progression. Therefore, we focused on exosomes derived from gastric cancer cells, the action of gastric cancer cells, further constructed a gastric cancer peritoneal dissemination mouse model, the action of exosomes in gastric cancer peritoneal metastasis mechanism was examined in vitro, in vivo, respectively.

Method: Exosome Isolation Kit was used to extract exosomes from each gastric cancer cell line MKN-1, MKN-45, MKN-45 GFP. The determination of exosomes was carried out using Nano-drop®. Cell adhesion in exosome-treated and non-administered groups were analyzed by colony forming assay. Cell proliferation ability was analyzed using cell proliferation assay method. In vivo, we examined whether the degree of peritoneal dissemination was made using live cell imaging using fluorescent cells. MKN-45 GFP was intraperitoneally administered to BALB/c-nu mice. On days 0, 5, 10, 15, 20, groups were prepared in which 25 µg/500 µl of exosome was administered intraperitoneally and groups in which the same amount of PBS was administered intraperitoneally. In addition, MKN-45 GFP was subcutaneously injected into BALB/c-nu mice to create MKN-45 GFP tumors, which were divided into small horns and transplanted to the stomach walls of BALB/c-nu mice. In the same way as the previous method, we prepared a group in which 25 µg/500 µl exosome was intraperitoneally administered on days 0, 5, 10, 15, 20 and a group in which the same amount of PBS was intraperitoneally administered. We performed a visual comparison of transplanted cancer cells and peritoneal dissemination of cancer.

Results and Discussion: The results of the colony forming assay method suggest that exosomes derived from gastric cancer cells have the effect of promoting colony formation ability of cancer cells. We evaluate the results of peritoneal seeding model mice in this study, and further proceed with the analysis of miRNA extraction and analysis from exosomes derived from gastric cancer cells, and cell adhesion of cancer cells under high concentrations of exosomes.

Citation Format: Kazuya Kinoshita. The relationship between peritoneal dissemination of gastric cancer & exosome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2905.
Abstract 2939: Activation of NRF2 signaling accelerates development of small cell lung cancer in mice
Authors: Hamad Samera , Montgomery Stephanie , Bowman Brittany , Murphy Ryan , Randell Scott , Oliver Trudy , Hayes D. Neil , Major M. Ben , Weissman Bernard
Published: July 2021

Activating mutations in NRF2 frequently occur in human lung cancers, especially the E79Q mutation. However, how the activating mutation contributes to lung tumor development is not well understood. We used a new genetically engineered mouse model (GEMM), LSL-Nrf2E79Q/+ with Cre-inducible expression of this mutation within the endogenous Nrf2 locus, to understand its contribution to lung tumor development. We compared tumor development in p53fl/fl;p16fl/fl;LSL-Nrf2+/+ (WT) versus p53fl/fl;p16fl/fl;Nrf2E79Q/+ (Het) mice after intranasal instillation of Adenoviral Cre. While we did not observe a significant difference in lung adenocarcinoma (LUAC) between the Het and WT mice, the Het mice showed significantly higher frequencies of bronchial neuroendocrine tumors that resembled small cell lung carcinomas (SCLC) compared to WT. Additionally, the latency of SCLC lesions was lower in Het mice compared to WT. Surprisingly, LUACs in both groups were negative for NRF2 by IHC labeling despite recombination of the mutant Nrf2 allele. While all lesions in the WT and Het mice displayed positive ASCL1 and CGRp (NE markers) IHC labeling, only the SCLCs in Het mice expressed NRF2. This is the first study showing activation of Nrf2E79Q mutation drives development of small cell lung carcinomas. Interestingly, our data also suggest that the E79Q NRF2 activating mutation may inhibit the progression of LUAD.

Citation Format: Samera Hamad, Stephanie Montgomery, Brittany Bowman, Ryan Murphy, Scott Randell, Trudy Oliver, D. Neil Hayes, M. Ben Major, Bernard Weissman. Activation of NRF2 signaling accelerates development of small cell lung cancer in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2939.
Abstract 2942: Improved LAG-3 humanized knock-in mouse model for assessment of mono- and combination therapy strategies for cancer treatment
Authors: Li Huilin , Zhou Xiaofei , Li Dirui , Chromikova Veronika , Lin Qingcong
Published: July 2021

In recent years, immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment. One of the potential therapeutic targets that seems increasingly attractive is LAG-3 (Lymphocyte activation gene 3, CD223), a human protein encoded by the LAG3 gene. LAG-3 can be found on activated T cells, NK cells, B cells and plasmacytoid DCs and serves as a negative regulator of immunity. Inhibition of LAG-3 enhances the antitumor effect of specific CD8+ T cells, which is an observation pursued by several pharmaceutical companies with their products in pre-clinical or early clinical development. Therefore, it is clear there is a need for suitable pre-clinical models able to accurately reflect the relevant physiological processes to evaluate the safety and efficacy of LAG3-targeted therapeutics. Biocytogen has generated a humanized LAG-3 mouse model for both in vitro functional validation and in vivo efficacy evaluation of anti-hLAG-3 antibodies. The targeting strategy is that exons 2~7 of mouse Lag3 gene which encode the extracellular domain are replaced by human LAG3 exons 2~7. After an introduction of this modification, human LAG-3 was detectable on Tregs in the mouse spleen and the anti-hLAG-3 antibodies and recombinant human FGL-1 protein associated well to the mouse splenocytes. Basal leukocyte subpopulations, including T/B/NK cells, DC, granulocytes, and monocytes/macrophages, were similar between humanized and wild-type mice. Additionally, anti-human LAG-3 antibodies partially blocked LAG-3/FGL-1 binding. Furthermore, using the MC38 tumor model, we show robust efficacy of anti-human LAG-3 antibodies in inhibiting tumor growth in vivo.In summary, LAG-3 humanized mice are a useful tool for in vivo efficacy evaluation of anti-human LAG-3 antibodies. Moreover, Biocytogen has also generated PD-1/PD-L1/LAG3 multi-gene humanized mice, in which blood cell composition, morphology and the levels of ALT, AST and other indicators were comparable to wild-type mice. This model is ideal to assess an increased therapeutic benefit in cancer treatment provided by combination therapies. Indeed, in our hands using the MC38 tumor model, the combination of anti-LAG-3 antibodies with anti-PD-1 (or anti-PD-L1) antibodies shows synergistic inhibitory effects , demonstrating that the PD-1/PD-L1/LAG3 humanized mice represent a powerful tool for preclinical assessment of in vivo efficacy of future therapeutics.

Citation Format: Huilin Li, Xiaofei Zhou, Dirui Li, Veronika Chromikova, Qingcong Lin. Improved LAG-3 humanized knock-in mouse model for assessment of mono- and combination therapy strategies for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2942.
Abstract 295: Implementing fully automated kinase inhibitor characterization using a robotic system
Authors: Thomas Daniel A. , Fleming Tom A. , Bittner Martin-Immanuel
Published: July 2021

About 50 kinase inhibitors have been approved by the FDA for different indications so far. On the way from target validation to approval, the biochemical characterisation of a novel kinase inhibitor is a challenging, yet absolutely critical task. High resolution, kinetic molecular profiling can enable better data-driven decision making early on in the drug discovery process, not only saving time and resources, but also leading to superior molecular design. Arctoris developed a robotics-enabled process for fully automated kinase inhibitor characterisation, providing an unparalleled depth of data capture, going beyond the current state-of-the-art of biochemical assay setup. We validated our technology platform establishing assays against four members of the Janus Kinase family (JAK1, JAK2, JAK3, TYK2), profiling a set of JAK inhibitors. Of note, several JAK inhibitors with prior FDA approval for other indications entered clinical trials for COVID-19 treatment, making this target class particularly relevant for an in-depth study. Reagent validation, assay development, calibration, and optimization were expedited through systematic multifactorial experimental design, high density assay plate formats and versatile automated liquid handling. The Arctoris Ulysses platform affords 9 orders of magnitude range in liquid volume handling, with picolitre precision and contact-free digital dispensing for true, non-serial, independent experimentation. Fully automated protocols were optimized, validated, versioned, and explicitly encoded. Robust potency measurements of all inhibitors were established against each of the JAK targets, revealing molecules with distinct isoform selectivity. Designing and selecting molecules with specific activity profiles enables the fine tuning of pharmacology and the avoidance of unwanted off-target toxicity. Our unique platform, assay design, and deep expertise enabled the identification of molecules within the JAK inhibitor set that exhibit a range of kinetic properties. Our mechanistic analyses can help to elucidate the mode of inhibition (competitive, allosteric, synergistic etc.) as well as provide information pertaining to the kinetic selectivity that may be present but missed by focusing solely on potency.

Citation Format: Daniel A. Thomas, Tom A. Fleming, Martin-Immanuel Bittner. Implementing fully automated kinase inhibitor characterization using a robotic system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 295.
Abstract 2959: Establishment of KRAS-G12C inhibitor induced resistant tumor models enable the development of new generation KRAS-G12C inhibitors and combinatorial strategies
Authors: Liu Gaoxiang , Shi Wenting , Gu Qingyang , Ji Qunsheng
Published: July 2021

The KRAS (Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) gene is one of the most popular oncogenes in the recent targeted therapy research. Activating KRAS mutations including G12C, G12D, etc are detected in around 25% of human cancers. The most common KRAS mutation is G12C, which comprises nearly 15% of lung adenocarcinoma, 8% of colorectal carcinoma and 4% of pancreatic adenocarcinoma. AMG-510 from Amgen and MRTX849 from Mirati Therapeutics both targeting KRASG12C mutant have shown encouraging activities in early clinical trials, especially shown a better overall response rate (ORR) and disease control rate (DCR) in KRASG12C mutant non-small cell lung cancer (NSCLC). However, acquired resistance to KRAS-G12C inhibitors will occur after prolonged treatment, which limits the effective evaluation in clinical trials. Various combinatorial strategies have been tried to overcome the resistance to KRAS-G12C inhibitors, including co-targeting vertical Ras signaling pathway and combining KRAS-G12C inhibitors with chemotherapeutics or immunotherapies. In order to facilitate the development of KRAS-G12C inhibitors, we have established an AMG510 resistant MIA PaCa-2 pancreatic adenocarcinoma CDX model and several colorectal carcinoma PDX models. These resistant models were derived from sensitive models carrying KRASG12C mutation, by prolonged treatment of AMG510 during several passages until a stable drug resistance phenotype occurred. To explore the resistant mechanisms, we performed Western blotting on downstream effectors ERK and found AMG510 produced less inhibition effect on ERK phosphorylation level in the resistant models. We are also developing more CDX and PDX models which are resistant to KRAS-G12C inhibitors. Therefore these models can be used to simulate acquired resistance to KRAS-G12C inhibitors after prolonged treatment in clinic, and provide a new tool to evaluate new generation of KRAS-G12C inhibitors as well as the combinatorial strategies with KRAS-G12C inhibitors.

Citation Format: Gaoxiang Liu, Wenting Shi, Qingyang Gu, Qunsheng Ji. Establishment of KRAS-G12C inhibitor induced resistant tumor models enable the development of new generation KRAS-G12C inhibitors and combinatorial strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2959.
Abstract 2963: A novel uterine leiomyosarcoma mouse model
Authors: Ng Chun Wai , Wong Kwong-Kwok
Published: July 2021

leiomyosarcoma is an extremely rare but clinically aggressive uterine cancer arising from the smooth muscle of the uterus. Each year, it is estimated to occur in 6 out of every 1,000,000 women in the United States. The average age at diagnosis is 51 and women with uterine leiomyosarcomas have a poor prognosis. Current therapies for uterine leiomyosarcoma are not very effective and the primary treatment of uterine leiomyosarcoma is surgery. Due to the intrinsic rarity of this disease, prospective randomized clinical trials examining the outcome in individuals with uterine leiomyosarcoma is limited. Moreover, a poor understanding of the pathogenesis of this disease is also a major knowledge gap to develop effective treatment strategies. Thus, a preclinical uterine leiomyosarcoma models that recapitulate the human disease is desperately needed to support clinical trial readiness. Since both p53 mutations and Pten homozygous deletion have been observed in 61% and 64% human uterine leiomyosarcomas respectively [3], we are generating genetic mouse models with Amhr2-Cre Ptenfl/fl p53R245Wfl/fl (homozygous p53 mutation), Amhr2-Cre Ptenfl/fl p53R245Wfl/+ (heterozgyous p53 mutation) and Amhr2-Cre Ptenfl/fl (control) genotypes. Our unpublished preliminary data showed that mouse with homozygous p53 mutation and homozygous deletion of Pten developed metastatic uterine leiomyosarcoma at 24 weeks of age with 100% penetrance. However, mouse with heterozygous p53 mutation and homozygous deletion of Pten did not develop uterine leiomyosarcoma at the age of 24 weeks. From the public available human uterine leiomyosarcoma RNAseq data, 80% of those tumors with p53 mutations had homozygous mutations. Previous mouse model with homozygous deletion of p53 gene and/or BRCA1 with Amhr2-Cre only had uterine leiomyosarcoma developed in approximately 50% of mice around 13 months without metastasis.

RNAseq and RPPA functional data of the uterine leiomyosarcomas from our novel mouse models are being generated for comparison with known genomic profiles of human uterine leiomyosarcomas. The RNAseq and RPPA functional proteomic data from comparing uterine tissues from homozygous and heterozygous p53 mutated mice provide insight in the understanding the development of uterine leiomyosarcoma and for future clinical trials.

Citation Format: Chun Wai Ng, Kwong-Kwok Wong. A novel uterine leiomyosarcoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2963.
Abstract 2973: Establishment of organoids and patient derived cancer cell lines from gastric cancer body fluids as preclinical models for personalized therapy
Authors: Kang Sun Kyoung , Bae Hyun Joo , Kwon Woo Sun , Kim Tae Soo , Lee Sujin , Cho Sang Woo , Chung Hyun Cheol , Rha Sun Young
Published: July 2021

Gastric cancer (GC), the second leading cause of cancer-related deaths worldwide, is especially prevalent in Asian countries. Despite advances in therapeutics, GC patients with peritoneal metastasis is frequent and when ascites has developed, the prognosis is very poor. Also, the number of anticancer drugs successfully marketed is rare in GC. Therefore, a faithfully in vitro model that can test various drugs and perform patient-specific personalized therapy is needed. We isolated cancer cells from ascites or pleural fluids of gastric cancer patients and established 19 patient-derived organoids and matched patient-derived cancer cell lines (PDCC). Organoid success rate was 60.7 % while PDCC was about 28.5 %. The time required to establish organoids was 1-2 months, while PDCC was 3-4 months. Organoids showed various growth characteristics and morphology, which reflects the clinical behavior of the patients including adenocarcinoma or signet ring cell carcinoma. In addition, drug-sensitive to each organoid and matched PDCC were identified using various chemotherapeutics and targeted agents. Then, patients' clinical information and comparative analysis were performed, suggesting the potential sensitive drug to each patient. Currently, the genetic information of each organoid is ongoing with WES/RNA Seq, and the sensitivity and comparative analysis of the drug will be performed. Therefore, our results provide a potential infrastructure for personalized therapy by comprehensive analysis of the patient's genomic data and organoid/PDCC model.

Citation Format: Sun Kyoung Kang, Hyun Joo Bae, Woo Sun Kwon, Tae Soo Kim, Sujin Lee, Sang Woo Cho, Hyun Cheol Chung, Sun Young Rha. Establishment of organoids and patient derived cancer cell lines from gastric cancer body fluids as preclinical models for personalized therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2973.
Abstract 2975: Genome-scale CRISPR screen finds prostate lineage specific dependencies
Authors: Attari Adel , Misek Sean , Eser Pinar , Yeagley Alexa , Chen Yu , Boehm Jesse , (Moony) Tseng Yuen-Yi
Published: July 2021

Functional genomics has held great promise for mapping cancer dependencies and identifying new therapeutic targets in cancer cell lines. However, this remains a major challenge for prostate cancer (PCa) due to the lack of cancer cell models and efficient optimization of pooled genetic perturbation screens. The Broad Cancer Cell Line Factory (CCLF) is attempting to fulfill this unmet need by generating novel prostate cancer cell models and uncovering prostate specific lineage dependencies. Encouragingly, Dr. Yu Chen at Memorial Sloan Kettering Cancer Center has developed several 3D cancer organoids from advanced prostate cancer. Although the success rate needs improvement for primary PCa, over 12 long-term models have been established by this group. In collaboration with Chen Lab, we aim to expand the prostate cell model collection by using PCa organoids and patient-derived normal cells. We can then apply these models for genetic perturbation profiling to unravel prostate lineage specific dependencies. Many historical cancer cell lines and cancer organoids were not compatible with genome-scale CRISPR screening due to the slow cellular growth. Here we first optimized the media conditions in these PCa organoids by using empirical media screening assay conditions through our HYBRID technology. The HYBRID technology allows for the systemic evaluation of 64 media conditions at once, so that we can perform RNAseq analysis to identify the conditions most physiologically resembling the PCa environment with the maximum doubling time. We have further developed our high throughput screening strategies for organoid culture using an optimized protocol to study 3D growth patterns in a 96-well format using the Incucyte S3 System. Next, to map out all possible dependencies, we are testing the feasibility of a pooled genome scale CRISPR screen. In our preliminary assay development results, we demonstrated the reproducible viral infectibility in several organoids ranging from 40-70% infection rate. We anticipate that these genome-wide CRISPR data in PCa organoid and normal prostate cell models can be integrated and analyzed with 4 previous PCa cell lines screened in the Broad Dependency Map to point out more prostate lineage specific genetic vulnerabilities and possible therapeutic targets.

Citation Format: Adel Attari, Sean Misek, Pinar Eser, Alexa Yeagley, Yu Chen, Jesse Boehm, Yuen-Yi (Moony) Tseng. Genome-scale CRISPR screen finds prostate lineage specific dependencies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2975.
Abstract 3: Deep learning-based integration of esophageal cancer morphology with genomics
Authors: Verma Ruchika , Wu Wei , Kumar Neeraj , Yu Elizabeth , Choi Won-Tak , Umetsu Sarah , Bivona Trever
Published: July 2021

Esophageal cancer is a major cause of cancer mortality world-wide. Even with advanced treatment options available, patients with esophageal cancer have a poor 5-year survival rate of 16%. Genomic studies of esophageal cancer have revealed frequently mutated genes involved in different oncogenic pathways. In this study, we attempted to predict genomic alterations, pathological and clinical features directly from H&E-stained whole slide images (WSIs) of individual esophageal cancer cases by utilizing modern deep learning methods. We used WSIs with esophageal cancer obtained from TCGA and annotated cancerous epithelial and stromal components separately for esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) (n= 25 cases for each) to create a training set. Subsequently, patches of size 256×256 pixels were extracted, and a U-Net convolutional neural network (CNN) was trained for semantic segmentation of tumor epithelium and stromal pixels. Tumor epithelium regions identified by the first U-Net CNN were fed into a Squeezenet model to learn to distinguish between EAC and ESCC in the training phase. Finally, for each subtype (EAC and ESCC), separate Densenet models were trained to predict gene mutation status. We also trained independent Densenet models to predict tumor stage, grade, and survival outcome from the tumor epithelial patches. After training, we tested these models on an independent validation set obtained from TCGA (n = 99) and UCSF (n = 25) patients, ensuring patient level separation between the training and the validation sets. We obtained an average AUC of 0.90 and 0.87 for epithelium vs. stromal and EAC vs. ESCC classification, respectively, on the validation datasets. These models enabled us to predict tumor stage, grade, and survival outcome with an accuracy of > 0.9. TP53 gene alterations were detected in over 50% of esophageal cancer cases, and our model was able to predict TP53 gene mutation status on WSIs with an AUC of 0.91. Overall, this study demonstrates how deep learning algorithms can aid in better understanding and predicting key biological and clinical features of aggressive malignancies at both population and individual patient level. This integration of artificial intelligence with molecular and histopathologic features could also help to accelerate precision cancer diagnosis and treatment in the clinical setting.

Citation Format: Ruchika Verma, Wei Wu, Neeraj Kumar, Elizabeth Yu, Won-Tak Choi, Sarah Umetsu, Trever Bivona. Deep learning-based integration of esophageal cancer morphology with genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3.
Abstract 3003: In vitro and in vivo model for evaluation of novel therapeutic agents in patient -derived acute B-lymphoblastic leukemia (B-ALL)
Authors: Vishwakarma Bandana Ajay , Verma Bhavna , Wesa Amy
Published: July 2021

B-cell Acute Lymphoblastic Leukemia is a hematological malignancy with malignant transformation and proliferation of B-Cell progenitors. Young patients fare well with common therapeutic agents and hematopoietic stem cell transplant, but a subpopulation is chemo-resistant and has high relapse rate. Besides, prognosis in adult patient is worse than younger individuals. Thus, there is an increase need for development of novel and targeted therapeutics. Lack of suitable in vitro culture conditions for primary B-ALL cells severely impairs the evaluation of new therapeutics. We have established a short-term culture system that supports survival of primary B-ALL cells ex vivo for screening of candidate therapeutic agents. Different culture conditions were evaluated, and cell growth/viability was assessed using Cell titer-Glo assay. Out of 13 primary B-ALL models studied 5 models proliferated and 5 remain stable and viable till day 3. Flow cytometry study showed that all the models stably expressed CD19 and CD20 throughout the assay. The models were tested for sensitivity to chemotherapeutic drugs doxorubicin, vincristine, imatinib and cytarabine. Heterogeneous dose dependent responses were observed across the models (IC50 17nM - 0.3 µM doxorubicin), (IC50 3 - 79 nM vincristine) indicating a range of relative sensitivity to B-ALL models. Interestingly, BCR-ABL-ve models were resistant to imatinib and BCR-ABL+ve sensitive. Next ,to develop patient derived xenograft preclinical B-ALL model, two million PBMC from B-ALL patients (n=14) were intravenously transplanted into sub-lethally irradiated NCG mice. 100% engraftment(n=5,P1) was observed in all the models, but latency of engraftment(>3%) in peripheral blood varied from 15 days to 3.5 months. The time of passage varied from 40 to 120 days. Flow cytometry analyses showed 30-95% of the bone marrow cells was hCD45+, hCD19+ cells. Splenomegaly was observed and infiltration of 40-98% of B-ALL cells was detected in spleen. Engrafted cells matched the immunophenotype of the corresponding patient. On serial transplantation (n=15) these models showed similar engraftment and immunophenotype. Thus, at Champions we have established a robust in vitro platform for screening new drugs for B-ALL and a passagable patient derived preclinical xenograft model for in vivo drug efficacy study.

Citation Format: Bandana Ajay Vishwakarma, Bhavna Verma, Amy Wesa. In vitro and in vivo model for evaluation of novel therapeutic agents in patient -derived acute B-lymphoblastic leukemia (B-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3003.