bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 321: Novel bone microenvironment model of prostate cancer with chitosan fiber matrix and osteoblast in 3D culture
Authors: Samoto Masahiro , Matsumoto Hiroaki , Hirata Hiroshi , Ozawa Sho , Mori Junichi , Inoue Ryo , Yano Seiji , Yamamoto Yoshiaki , Matsuyama Hideyasu
Published: September 2020

BACKGROUND: The interaction between prostate cancer cells and osteoblast is essential for the development of bone metastasis. Recently, novel androgen receptor signal inhibitors (ARSi) have been approved for metastatic castration naïve (mCNPC), or non-metastatic castration resistant prostate cancer (nmCRPC), both of which should be pivotal to investigate the association between bone microenvironment and tumor. We established a novel 3D in vitro culture method reflecting bone microenvironment and evaluated the drug susceptibility of ARSi including enzalutamide, apalutamide, darolutamide, and abiraterone (Abi) with/without dutasteride (Duta).

METHODS: GFP-transferred C4-2 (CRPC cell line) and RFP-transferred human osteoblast differentiated from human-mesenchymal stem cell was co-cultured in chitosan nanofiber, a mimicry of 3D scaffold of bone microenvironment, coated culture plate (Cosmo bio Co., LTD). The growth of C4-2 cell was quantified using live-cell imaging and analysis system, Cell3 iMager duos (SCREEN). Abi metabolites including delta-4 abiraterone (D4A) and 3-keto-5-alfa abiraterone in C4-2 cells were measured by ESI-TOF-MS method.

RESULTS: We could non-invasively quantify the sustained growth of C4-2 cells at maximum of 30 days. IC50 of each drugs and combination effect of Abi and Duta was evaluated using this model. Combination treatment synergistically inhibited the growth of C2-4 cells compared with each drug alone. D4A, the strongest anti-proliferative metabolite of Abi under the CRPC-specific backdoor pathway, was detected from the treated C4-2 cell. These results were coincided with the effective cases of pharmacokinetics of Abi metabolites in our ongoing phase II clinical trial of combination therapy of Abi with Duta for CRPC (UMIN000027795). Furthermore, combination of abiraterone and dutasteride was most potent cell growth inhibitor than other ARis tested in this model. This model is applying for international patent (C12N 5/09).

CONCLUSIONS: Our bone microenvironment model is unique and useful to evaluate the new drug susceptibility testing in prostate cancer cells. This model may help in disclose unknown mechanisms from micro- to clinical bone metastasis in prostate cancer.

Citation Format: Masahiro Samoto, Hiroaki Matsumoto, Hiroshi Hirata, Sho Ozawa, Junichi Mori, Ryo Inoue, Seiji Yano, Yoshiaki Yamamoto, Hideyasu Matsuyama. Novel bone microenvironment model of prostate cancer with chitosan fiber matrix and osteoblast in 3D culture [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 321.
Abstract 322: A unique ex vivo tumor model: 3D co-cultured system with cancer cells and stromal tissue for drug evaluation
Authors: Takahashi Yuki , Tsukamoto Kei , Shimizu Yuki , Katayama Ryohei , Shinozaki Eiji , Nagayama Satoshi , Matsusaki Michiya , Kitano Shiro , Yamaguchi Kensei , Fujita Naoya
Published: September 2020

Background;Recently, three-dimensional culture model has been reported as better tumor model than 2D culture. The importance of interaction between cancer and stromal cells has been widely recognized in tumor progression and resistance against treatment. So that ex vivo model which recaptures the tumor microenvironment is required to perform drug evaluation mimicking the patient tumor tissue.

Methods; Layered 3D stromal tissues with microvascular network were produced by culturing normal fibroblasts and endothelial cells coated with the extra-cellular matrix (ECM) and natural polysaccharide, namely collagen and heparin. The layered 3D stromal tissues and co-cultured tumor were morphologically evaluated by HE staining, immunohistochemistry and immunofluorescence (IF). Drug sensitivity assay were conducted using popular colorectal cancer cell lines, and patient-derived cell lines (PDCs) established in JFCR under approved IRB. Remaining cancer cells post drug treatment were quantified by IF and imaging analysis. Furthermore, in vivo drug sensitivity was also evaluated.

Results: The CD31 positive luminal structures which shows vascular endothelial tube formation were observed intra multi-layered tissues. The dense microvascular network tended to be formed nearby cancer cells. In comparison with 2D culture model, decreased drug sensitivities were represented in the layered 3D co-cultured model. The evaluated drug sensitivities in 3D model may reflect the response of cancer cells in in vivo.

Conclusion; We developed the layered 3D stromal tissue culture system including blood micro-vessels. Our unique 3D ex vivo model appear to be a valuable tool for drug evaluation, and thus further studies are underway to confirm the usefulness of our model. Testing approved and/or developing compounds with PDCs in the model would potentially enable better prediction their efficacy.

Citation Format: Yuki Takahashi, Kei Tsukamoto, Yuki Shimizu, Ryohei Katayama, Eiji Shinozaki, Satoshi Nagayama, Michiya Matsusaki, Shiro Kitano, Kensei Yamaguchi, Naoya Fujita. A unique ex vivo tumor model: 3D co-cultured system with cancer cells and stromal tissue for drug evaluation [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 322.
Abstract 3226: Facilitating personalized medicine with cloud-based storage and analytics
Authors: Howard Rachel , Hicks Kevin , Teer Jamie , Reisman Phillip , O'Leary Mandy , Eschrich Steven , Mitchell Ross , McLeod Howard , Rollison Dana
Published: September 2020

The vast wealth of medical data collected over the last decade holds great promise for accelerating novel research, discovery, and clinical translation. Specifically, the rapid expansion of genomic testing provides new opportunities for the clinical management of cancer patients, influencing diagnosis, risk stratification, and treatment planning. Moffitt Cancer Center's Personalized Medicine Clinical Service integrates next-generation sequencing test results into patient care, using the data to guide individualized treatment plans. To maximize the efficiency and efficacy of this service, creative solutions for data harmonization, storage, and management are required. We implemented a commercial molecular data warehouse (MDW), directly linked to our existing clinical data warehouse, to store and manage molecular data ranging from genotypic alterations to annotations from public resources (HUGO, COSMIC, Ensembl) and clinically actionable targets (4,256 records currently loaded). A centralized, cloud-based data and analytics platform is also being implemented at Moffitt that will integrate a broad range of multi-modal data. In the cloud environment, the data from the MDW will be linked to typically siloed data streams from the electronic health record, cancer registry management system, biospecimen management system, billing and scheduling systems, patient-reported information and outcomes, and patient-generated health data, creating a unique and customized Personalized Medicine Curated Data Mart (CDM). In addition to describing the features of the MDW and the challenges faced during its implementation, we will provide an overview of the extensive data cleaning and curation required to facilitate such a CDM. This includes the extraction of disease characteristics from unstructured clinical text via natural language processing, creation of new derived data fields, approaches to extracting and managing complex treatment data, and the inclusion of detailed, manually-abstracted recurrence and outcomes data for historical patients from existing institutional datasets such as the Clinical Genomics Action Committee (CGAC) database. Finally, we will present prototypes of analytics dashboards that will interface directly with our CDM, facilitating intuitive data exploration for all members of our personalized medicine teams.

Citation Format: Rachel Howard, Kevin Hicks, Jamie Teer, Phillip Reisman, Mandy O'Leary, Steven Eschrich, Ross Mitchell, Howard McLeod, Dana Rollison. Facilitating personalized medicine with cloud-based storage and analytics [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 3226.
Abstract 3258: Reconstituted functional Tim-3/Gal-9 immune checkpoint cell line to screen for novel Tim-3 ICBs
Authors: Zhang Fang , Wu Pei , Liu Jun , Wang Baiqiu
Published: September 2020

Background: Tim-3 (T-cell immunoglobin and mucin-domain containing-3) has emerged as a promising novel immune checkpoint target playing non-redundant and synergistic roles with PD-1 in triggering T cell exhaustion, however, the complexity of Tim-3-interacting ligands (Gal-9, Ceacam1 and others) has posed a challenge to reconstitute functional Tim-3-targeting immune checkpoint blockers (ICBs) cellular screening system.

Materials and methods: To address this challenge, we have engineered Tim-3 Advanced Genomic Manipulated human Jurkat T IL-2-Luc2P reporter cells (Tim-3 AGM T cells) via multiple steps of electroporation-mediated genomic manipulation, and validated the functionality of this reconstituted Tim-3 immune checkpoint cellular system using IL-2-Luc2P reporter assay and IL-2 ELISA following TCR and CD28 engagement.

Results: We found that anti-CD3/CD28-induced T cell activation in the Tim-3 AGM T cells as assessed by IL-2-Luc2P reporter was significantly suppressed (~80%) after over-expression of Gal-9 but not the empty vector control, validating functionality of the reconstituted Tim-3/Gal-9 immune checkpoint complex.

Conclusions: Such an innovative reconstituted functional Tim-3/Gal-9 immune checkpoint reporter cell line can enable high through-put screening for novel Tim-3 ICBs including small or large molecule therapeutics and following in-depth mechanistic studies.

Citation Format: Fang Zhang, Pei Wu, Jun Liu, Baiqiu Wang. Reconstituted functional Tim-3/Gal-9 immune checkpoint cell line to screen for novel Tim-3 ICBs [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 3258.
Abstract 3268: Anti-tumor effect of VSIG4 in conversion of immunosuppressive type II macrophage via repolarization
Authors: Kim Hye Jeong , Hwang Sunhee , Lee Seung-Hyun , Son HyunTae , Lee Joong Won , Kwon Byoung S.
Published: September 2020

V-set and immunoglobulin domain-containing 4 (VSIG4) is a B7 family-related protein which includes PDL1, VISTA and CTLA4 ligand. The known functions of VSIG4 are a receptor for complement iC3b and C3b to inhibit complement activity and negative regulation of proliferating T cell. Although VSIG4 is highly expressed in tissue-resident macrophage and tumor-associated macrophage (TAM), the mechanism of the signal transduction is not fully elucidated. Here we investigate the pivotal roles of anti-VSIG4 therapeutic antibody in TAM and tumor microenvironment. Anti-VSIG4 antibody engagement altered a broad range of type II macrophage (M2) transcriptome which related to immune-tolerance and increased the proliferation of cytotoxic CD8+T cells. Anti-VSIG4 antibody induced phosphorylation of JNK and p38MAPK and in consequence, decreased the expression of M2 marker CD14, CSF-1R and CD163 while type I macrophage (M1) marker CD86 and CD40 were increased. Furthermore, anti-VSIG4 antibody treatment to TAM in ovarian cancer patients, results in secretion of inflammatory cytokines and chemokines in a similar way to M1 macrophages. In the CD34+-based humanized mouse model, stimulation of VSIG4 attenuated tumor growth and promoted infiltration of IFN-gamma-producing CD8+ T cells into tumor tissue. Adopted transfer of differentiated M2 macrophages to immune-deficient NSG mice confirmed M2 conversion into M1 macrophage phenotype with anti-VSIG4 antibody. Our finding suggests that anti-VSIG4 antibody directly act on TAM and induce repolarization of TAM to tumor suppressive M1 macrophages, leading to a CD8+ T cell proliferation and tumor suppression. Overall, targeting the VSIG4 would be an advantageous candidate in cancer therapeutics.

Citation Format: Hye Jeong Kim, Sunhee Hwang, Seung-Hyun Lee, HyunTae Son, Joong Won Lee, Byoung S. Kwon. Anti-tumor effect of VSIG4 in conversion of immunosuppressive type II macrophage via repolarization [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 3268.
Abstract 3289: The status of the tumor microenvironment changes dynamics of the balance of CD8+ T cells and Treg cells in renal cell carcinoma
Authors: Sugiyama Daisuke , Muramatsu Tomoaki , Kobayashi Yoichi , Sassa Naoto , Maruyama Shoichi , Goto Momokazu , Akatsuka Yoshiki , Nishikawa Hiroyoshi
Published: September 2020

Immune checkpoint inhibitors (ICIs) are widely applied into the clinic based on their remarkable clinical effectiveness. Tumor mutation burden (TMB) and PD-L1 expression in the tumor microenvironment (TME) have been shown to be correlated with clinical responses of ICIs. In contrast, immunosuppressive cells, especially regulatory T (Treg) cells, in the TME are thought to be associated with the resistance to ICI therapy. In this study, we investigated the role of Treg cells in the TME in renal cancer (RCC). We have previously reported that the RCCs can be classified into three groups based on the frequency of CD3+CD4+CD45RA+FoxP3highcells; effector/activated Treg (eTreg) cells: high: > 10%, moderate: > 5%, and low: > 0%, and high frequency of eTreg cells were correlated with the higher expression of PD-1, LAG-3, TIM-3 by CD8+ T cells. Based on the findings, we further investigated the frequency and phenotypes of Treg cells in the TME. We first compared flow cytometry analysis with immunohistochemistry analysis in the frequency of Treg cells. The frequency of Treg cells in the TME was well-correlated with FCM and IHC analyses. Then, the detailed phenotypes of tumor-infiltrating eTreg cells was examined. eTreg cells harbored high expression of activation markers such as TIGIT and CTLA-4, but low-moderate expression of exhausted molecules such as PD-1 and LAG-3. These data implicate that CD8+ T cells are exhausted, whereas Treg cells are activated in the TME. The clinical benefits by ICI therapy for RCC could be associated with the balance of CD8+ T cells and Treg cells. We are now analyzing the detailed mechanism controlling the balance to identify biomarkers of ICI therapy and to develop novel effective cancer immunotherapies.

Citation Format: Daisuke Sugiyama, Tomoaki Muramatsu, Yoichi Kobayashi, Naoto Sassa, Shoichi Maruyama, Momokazu Goto, Yoshiki Akatsuka, Hiroyoshi Nishikawa. The status of the tumor microenvironment changes dynamics of the balance of CD8+ T cells and Treg cells in renal cell carcinoma [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 3289.
Abstract 3308: SUMOylation-mediated enhancer activity regulates type I interferon pathway
Authors: Du Li , Chen Yuan , Rosen Steven
Published: September 2020

Type I interferon (IFN) pathway plays an important role in innate immunity and anti-tumor immune response. Previous study has reported that SUMOylation represses type I interferon production, and that deletion of the E2 enzyme Ubc9 or SUMO2 or 3 greatly enhanced type I interferon response. However, the detailed mechanism of how SUMOylation regulates type I interferon response in controversial in the literature. In this study, we found that inhibition of the SUMO E1 enzyme with ML-792 significant enhanced type I cytokine IFNβ expression in human monocytes THP1 cell line, up to 1,500 folds. Similar induction was observed in human peripheral blood mononuclear cells (PBMCs). However, ML-792 treatment showed no effect in IFNβ promoter reporter assay, indicating that ML-792 induced IFNβ expression is not through activation of the transcription factors, such as through the STING pathway, that act on the promoter region. Genome browser ENCODE analysis showed SET Domain Bifurcated Histone Lysine Methyltransferase 1 (SETDB1) binds at a ifnb1 enhancer region, known as L2, located ~9kb upstream of ifnb1 transcription start site (TSS). Knockdown of SETDB1 increased IFNβ level resulting in enhanced type I IFN downstream gene expression, indicating that SETDB1 is a transcriptional suppressor of ifnb1 gene. Chromatin immunoprecipitation (ChIP) assay determined that ML-792 treatment or knockdown of the catalytic subunit of the SUMO E1 greatly reduced SETDB1 SUMOylation and occupancy at ifnb1 gene enhancer region. Mutation of SETDB1 SUMOylation sites (4KR, with all 4 SUMOylation sites mutated: K355R, K1032R, K1050R, K1069R) also reduced SETDB1 binding at ifnb1 gene enhancer region shown by ChIP assay, demonstrating that SUMOylation is required for SETDB1 binding to ifnb1 gene enhancer region. In conclusion, our study identified SETDB1 as a novel transcriptional suppressor of ifnb1gene. In addition, we found that at least one mechanism of SUMOylation in preventing type I IFN production is through regulating SETDB1 binding to the enhancer of ifnb1 and consequently histone methylation.

Citation Format: Li Du, Yuan Chen, Steven Rosen. SUMOylation-mediated enhancer activity regulates type I interferon pathway [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 3308.
Abstract 331: Biology reconstructed: Testing immuno-oncology agents in tumor-specific 3D cell culture
Authors: Kirshner Julia , Parikh Mukti R.
Published: September 2020

In recent years, immuno-oncology had emerged as a promising strategy to control tumor growth and spread. However, the advancement of immuno-oncology and development of immuno-oncology agents are hindered by the lack of in vitro models capable of supporting an intact immune compartment. Here we describe a tumor-specific 3D cell culture platform where the human tumor microenviroment is reconstructed ex vivo. Our patient-derived 3D culture approach comprehensively accounts for the major components of the cellular and extracellular tissue microenvironment preserving the critical interactions between tumors and their surroundings. Our 3D tumor-spectific models incorporate tissue-specific extracellular matrix(ces) (ECM) and tumor-specific medium supplements allowing for a true reconstruction of tumor microenvironment. The system is complete with the immune along with tumor and stromal, elements embedded within the tumor-specific ECM. Our platform retains the characteristics of the in vivo tissue demonstrating long-term viability, proliferation, and maintenance of the cellular heterogeneity appearing within the native microenvironment. Our approach is compatible with multiple classes of immuno-oncology agents from naked antibodies and antibody-drug conjugates, to bi- and tri-specific compounds, and CAR-T cells. The experimental set-up for testing immuno-oncology agents is comprized of 3 step: (1) embed tumor, stromal, and immune cells within the tissue-specific ECM and overlay with growth medium; (2) add the treatment agents to the culture medium and allow to infiltrate the ECM; and (3) evaluate the effects of the test agents either in situ, by microscopy and/or cell-based assays, or after isolation from the ECM by FACs and omics approaches. The rate of agent infiltration into the ECM was dependent on the therapeutic modality and was observed within 24-96 hours of treatment. Measurable T cell activation was detected at 48 hours post treatment and anti-tumor effects were evident within 5-9 days of exposure to therapeutic agents. Importantly, activation of the T cells in response to treatment occurred without exogenous priming. Taken together, this data position our 3D platform as a superior alternative to the currently used methods for testing of immuno-oncology agents.

Citation Format: Julia Kirshner, Mukti R. Parikh. Biology reconstructed: Testing immuno-oncology agents in tumor-specific 3D cell culture [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 331.
Abstract 3338: Intra-tumoral microbes correlate with immune cell fractions in lung cancer biopsies not other cancers
Authors: Mo Xiaokui , Spakowicz Daniel , Hoyd Rebecca
Published: September 2020

Exogenous sequences in bacteria have been found in many tumors, but their effect on outcomes remains largely unknown. We hypothesize that intratumoral microbes elicit effects through the recruitment of immune cells, such as CD8+ T-cells that are important for immunotherapy response. Through ORIEN (Oncology Research Information Exchange Network), we obtained RNA-seq data from biopsies of 480 tumors (Melanoma=16, Bladder=104, Colorectal=20 Renal Cell Carcinoma=20, Sarcoma=118, Lung=202) from patients treated at The Ohio State University Comprehensive Cancer Center. Reads were aligned to human and exogenous genomes using TopHat2 and Kraken2/Bracken, respectively. Human gene expression was deconvolved to relative abundances of immune cells using CIBERSORT. Across cancers, an average of 99.87% of reads were aligned to the human reference genome. The immune cell types CD8+ T-cells, activated dendritic cells and M1 macrophages were significantly enriched in lung cancer (p-values <0.0001), and M2 macrophages were significantly enriched in sarcoma (p-values<0.0001). On the other hand, exogenous reads were aligned to 1328 genomes of bacteria, archaea, viruses and fungi. The phyla with the largest average relative abundances were Proteobacteria, Firmicutes and Actinobacteria, with average relative abundances of 39.5%, 20.1%, and 5.16% respectively. In lung cancer, there was a significant positive correlation between the fraction of CD8+ T-cells and Acinetobacter junii (p-value=0.035), and between activated NK cells and Acidipropionibacterium virtanenii (p-value=0.035). This suggests a mechanism by which intratumoral microbes may affect the immune system and thereby cancer outcomes.

Citation Format: Xiaokui Mo, Daniel Spakowicz, Rebecca Hoyd. Intra-tumoral microbes correlate with immune cell fractions in lung cancer biopsies not other cancers [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 3338.
Abstract 3344: The human commensal lactobacillus rhamnosus GG orchestrates an anti-tumor immune response
Authors: Owens Josh , Saeedi Bejan , Naudin Crystal , Hunter-Chang Sarah , Darby Trevor , Robinson Brian , Jones Rheinallt
Published: September 2020

Only 10% of patients with colorectal cancer (CRC) are approved for immunotherapy and only 50% of approved patients are responsive to this treatment. The 95% of CRC patients that are refractory to immunotherapy are so because of low anti-tumor immune responses in the gut mucosa. Mucosal immunity is sensitive to changes in the microbiome community structure, and to dietary supplementation with beneficial bacteria, known as ‘probiotics' which are known to augment mucosal immunity. Therefore, we supplemented the murine microbiome by daily oral gavage with the human commensal bacteria, Lactobacillus rhamnosus GG (LGG). We discovered that LGG increases colonic CD8 T cell responses by flow cytometry and immunofluorescence analysis. We did not believe that LGG was having a direct effect on CD8 T-cell function but was being augmented through an innate cell population. Using BATF3-null mice which lack conventional dendritic cells, we did not detect LGG-induced CD8 T-cell changes determining that this response is dendritic cell dependent. In addition, we sought to determine how LGG was being sensed by dendritic cells. Based off preliminary data and published reports, we utilized Toll like Receptor 2 (TLR2)-null mice and found that LGG-induced CD8 T cell induction also required TLR2 signaling. These data describe a novel mechanism whereby LGG induces colonic CD8 T cells activation through a dendritic cell and TLR2-dependent manner. As CD8 T-cells are critical in the anti-tumor response, we induced colonic tumors in mice by performing the DSS-AOM model. After tumors were established, we administered LGG daily by oral gavage. LGG supplementation resulted in significantly reduced tumor burden compared to control mice fed vehicle or a non-probiotic bacterium. Furthermore, this reduction in tumor burden was CD8 T cell dependent, as depletion of CD8 T cells abrogated this effect. In summary, oral supplementation with LGG may hold clinical promise in augmenting colonic anti-tumor immune responses and may increase immunotherapy response rates in patients.

Citation Format: Josh Owens, Bejan Saeedi, Crystal Naudin, Sarah Hunter-Chang, Trevor Darby, Brian Robinson, Rheinallt Jones. The human commensal lactobacillus rhamnosus GG orchestrates an anti-tumor immune response [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 3344.
Abstract 336: Organotypic co-cultures as a novel 3D model for head and neck squamous cell carcinoma
Authors: Thierauf Julia C. , Engelmann Luca , Laureano Natalia Koerich , Stark Hans-Jürgen , Prigge Elena-Sophie , Horn Dominik , Freier Kolja , Grabe Niels , Federspil Philippe , Plinkert Peter K. , von Knebel Doeberitz Magnus , Hess Jochen , Affolter Annette
Published: September 2020

Background. Head and neck squamous cell carcinomas (HNSCC) are histologically and molecularly heterogeneous tumors and frequently develop resistance mechanisms against treatment. Reliable patient-derived 3D tumor models are urgently needed to further study the complex pathogenesis of these tumors and how to overcome treatment failure.

Methods. We developed a 3D organotypic co-culture (OTC) model for HNSCCs that maintains the architecture and cell composition of the patients' tumor. A dermal equivalent, composed of healthy, human-derived fibroblasts embedded into viscose fibers, grows in an insert for approximately 10 days in culture and serves as a scaffold for the patient sample. After outgrowth of fibroblast within the dermal equivalent, we cultivated vital HNSCC tumor explants from 13 patients (non-HPV driven, n=7; HPV-driven HNSCC, n=6) by carefully placing them on top of the scaffold. Fractionated irradiation (IR) was applied to 5 samples. To evaluate tumor proliferation and areas of apoptosis, we performed immunohistochemistry and immunofluorescence with antibodies against ki-67, cleaved caspase-3, pan-cytokeratin (PanCK), p16INK4a, and vimentin.

Results. All 3D OTC models of non-HPV driven samples encompassed proliferative cancer cells during cultivation for up to 21 days. Proliferation indices of primary and 3D-OTC models were comparable and consistent over time. Overall, tumor explants displayed heterogeneous growth and invasion patterns. Radioresistant and clonally expanding tumor cells were enriched in 3D-OTC models after fractionated IR. HPV DNA was detectable in both primary and OTC (day 14) of HPV-driven tumors; However, p16INK4a expression levels were varying.

Conclusion. Our 3D-dimensional organotypic co-culture model for HNSCC supports cancer cell survival and proliferation in their original microenvironment. The model enables investigation of invasive cancer growth as well as clonal expansion of resistant cancer cells upon treatment. In the future, the introduced model might serve as a platform to perform sensitivity testing upon treatment to predict the patients' individual response to therapy.

Citation Format: Julia C. Thierauf, Luca Engelmann, Natalia Koerich Laureano, Hans-Jürgen Stark, Elena-Sophie Prigge, Dominik Horn, Kolja Freier, Niels Grabe, Philippe Federspil, Peter K. Plinkert, Magnus von Knebel Doeberitz, Jochen Hess, Annette Affolter. Organotypic co-cultures as a novel 3D model for head and neck squamous cell carcinoma [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 336.
Abstract 3368: A novel anti-CTLA-4 antibody with potent anti-tumor activity emerged using humanized CTLA-4 mouse model
Authors: Guo Chaoshe , Yang Yi , Li Zhihong , Li Yanan , Chen Yunyun
Published: September 2020

CTLA-4 (Cytotoxic T-lymphocyte antigen 4), also known as CD152, belongs to immunoglobulin superfamily. It is an intracellular protein expressed in resting T cells; CTLA-4 translocates to the cell surface and transmits an inhibitory signal to T cells. In addition, CTLA-4 competes with its homologous T-cell co-stimulatory protein CD28 for ligands expressed on APC, namely CD80 (B7-1) and CD86 (B7-2), thereby antagonizing CD28 and inhibiting CD28 signaling, resulting in inhibition of IL-2, IFN-γ, IL-4 cytokines production, IL-2 receptor expression and cell cycle progression. Because Tregs constitutively express CTLA-4 at high levels, anti-CTLA-4 therapy would be expected to have the greatest effect on these cells. Ipilimumab, a fully human IgG1 monoclonal antibody, is the only commercially available CTLA-4 antagonist. It has been approved by FDA for treatment of metastatic melanoma, adjuvant melanoma and advanced renal cell carcinoma, based on its significant effect of tumor suppression demonstrated in clinical trials.

We have interests in searching for potent CTLA-4 antagonists. We selected an antibody with the best efficacy, namely YH001, from the candidate molecules through Biocytoge's in vivo drug screening platform. YH001 is a recombinant humanized anti-CTLA-4 IgG1 monoclonal antibody and it can bind with human CTLA-4 potently and specifically, without reactivity with other CD28 family receptors. YH001 was able to block the CTLA-4/CD80 and CTLA-4/CD86 interactions and potentiate CD28 signaling in the presence of cognate TCR stimulation. Moreover, YH001 also induced stronger bioluminescence than ipilimumab in the reporter assay. YH001 showed more potent ADCC activities than ipilimumab. YH001 showed dose-dependent in vivo anti-cancer efficacy, as well as Treg depletion and increased CD8+/Treg ratio in human CTLA-4 knock-in mice bearing MC38 tumors.

Exposure to YH001 was dose proportional with a half-life of approximately 5 days in cynomolgus monkey. Our preclinical investigation of YH001 demonstrates Biocytogen's capability in discovering clinical candidates using its robust antibody discovery platform.

Citation Format: Chaoshe Guo, Yi Yang, Zhihong Li, Yanan Li, Yunyun Chen. A novel anti-CTLA-4 antibody with potent anti-tumor activity emerged using humanized CTLA-4 mouse model [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 3368.
Abstract 3369: Rapid screening of novel OX40 agonistic antibodies for cancer immunotherapy
Authors: Shen Yuelei , Li Zhihong , Yang Yi , Chen Yunyun , Zhu Yu
Published: September 2020

OX40, also known as TNFRSF4 or CD134, is a member of the tumor necrosis factor receptor superfamily and is an important co-stimulator of T cell responses. OX40L expressed at high levels on activated antigen presenting cells (APCs) can result in higher order multimerization of OX40 receptors, leading to stronger activation in T cells. OX40 plays an important role in memory T cell formation. The highest levels of OX40 are found on effector and central memory CD4+ T cells and regulatory T cells (Treg). Given the duality of OX40 agonism in both stimulating effector T cell activity and suppressing Treg activity, OX40 agonists have been widely explored in the setting of cancer immunotherapy.

We hoped to develop novel OX40 agonists that can effectively inhibit tumor growth. YH002 is a recombinant, humanized IgG1 monoclonal antibody that specifically targets OX40. In vitro studies demonstrated that YH002 bound specifically and potently to human and monkey OX40 proteins. YH002 was able to dose-dependently potentiate the activation of human OX40 on reporter cells in the presence of FcγRIIB-expressing accessory cells. YH002 mediated ADCC activity in vitro. YH002 demonstrated robust anti-tumor efficacy with a bell-shaped dose response relationship against established MC38 syngeneic tumors in human OX40 transgenic mice (B-hOX40 mice) and Treg depletion were noted in tumor-infiltrating lymphocyte analysis using the same murine model. In conclusion, YH002 enhances anti-tumor activities by stimulating effector T cells and overcoming immune suppressions via suppressing or depleting regulatory T cells. Furthermore, we showed that YH002 works in concert with Pembrolizumab in double humanized model of h-OX40 and h-PD1. The combination of YH002 and ipilimumab is synergistic in double humanized model of h-OX40 and h-CTLA4. In the GLP-compliant single-dose toxicology study, the MTD of a single i.v. dose of YH002 in cynomolgus monkeys was considered to be 200 mg/kg/day. Repeated i.v. infusions of YH002 to male and female cynomolgus monkeys at 10, 30, or 90 mg/kg for 29 days (QW×5) were tolerated, the HNSTD was considered to be 90 mg/kg.

Citation Format: Yuelei Shen, Zhihong Li, Yi Yang, Yunyun Chen, Yu Zhu. Rapid screening of novel OX40 agonistic antibodies for cancer immunotherapy [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 3369.
Abstract 340: ABI1 and androgen dependence in prostate cancer
Authors: Kotula Leszek , Porter Baylee A. , Li Xiang , Ortiz Maria A. , Khanna Rakesh , Zhang Fan , Kung Sonia H. , Fazli Ladan , Gleave Martin E. , Bratslavsky Gennady , Ross Jeffrey S.
Published: September 2020

Background: Androgen independence is the most challenging problem in the treatment of advanced prostate cancer. Although most patients with early-stage prostate cancer can be cured with surgical and/or radiation treatment, the remainder of patients ultimately recur with metastatic disease upon development of resistance to anti-androgen therapy. Epithelial mesenchymal transition (EMT) and lineage plasticity has been proposed as key mechanisms underlying resistance in treatment-induced prostate cancer with neuroendocrine features (NEPC). There has been a rise in treatment-induced NEPC with increased use of anti-androgen therapies. ABI1 regulates EMT by suppressing FYN-STAT3 activation downstream from noncanonical WNT pathway, but the interaction of ABI1 with androgen pathway is not known. We sought to determine the effect of neoadjuvant hormone treatment (NHT) on ABI1 expression levels in different tumor types.

Methods: Here we analyzed ABI1 expression (IHC) in tissue microarray containing 75 tumors with no prior treatment, 198 patients subjected to NHT prior to surgery and 39 castration resistant tumors (total n=307). Androgen-induced ABI1 expression was determined upon R1881 treatment by Western blotting and immunofluorescence in hormone- sensitive LNCaP cells.

Results: ABI1 expression was significantly downregulated in tumors from patients undergoing NHT(p<0.05). ABI1 expression was significantly increased in CRPC tumors developed while on hormone therapy compared to ABI1 expression in tumors undergoing NHT (p<0.001), or with NEPC (p<0.001). Consistently with these observations, ABI1 expression was upregulated upon treatment of LNCaP cells with R1881 and downregulated by treatment of cells with enzalutamide.

Conclusion: ABI1 is an androgen sensitive gene. ABI1 expression is increased in CRPC, most likely secondary to androgen receptor reactivation. It is subsequently downregulated in NEPC, hence, we hypothesize that this may be a key mechanism driving the NEPC phenotype through promoting EMT. Further understanding of the role ABI1 may play in this transformation will provide a more mechanistic understanding of prostate cancer progression and maylead to improved targeted treatment options.

Citation Format: Leszek Kotula, Baylee A. Porter, Xiang Li, Maria A. Ortiz, Rakesh Khanna, Fan Zhang, Sonia H. Kung, Ladan Fazli, Martin E. Gleave, Gennady Bratslavsky, Jeffrey S. Ross. ABI1 and androgen dependence in prostate cancer [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 340.
Abstract 3401: Targeting lysosomal homeostasis in ovarian cancer through drug repurposing
Authors: Marastoni Stefano , Pesic Aleksandra , Nair Sree Narayanan , Li Zhu Juan , Madani Ali , Haibe-Kains Benjamin , Wouters Bradly G. , Joshua Anthony
Published: September 2020

Background: Drug repurposing has become increasingly attractive as it avoids the long processes and costs associated with drug discovery. Itraconazole (Itra) is a broad-spectrum anti-fungal agent which has an established broad spectrum of activity in human cell lines including cholesterol antagonism and inhibition of Hedgehog and mTOR pathways. Many in vitro, in vivo and clinical studies have suggested anti-proliferative activity both alone and in combination with other chemotherapeutic agents, in particular in ovarian cancer. This study is aimed at supporting the therapeutic potential of Itra and discovering and repurposing new drugs that can increase Itra anticancer activity as well as identifying new targets in the treatment of ovarian cancer.

Methods: We tested a panel of 32 ovarian cancer cell lines with different doses of Itra and identified a subset of cells which showed significant sensitivity to the drug. To identify genetic vulnerabilities and find new therapeutic targets to combine with Itra, we performed a whole genome sensitivity CRISPR screen in 2 cell lines (TOV1946 and OVCAR5) treated with non-toxic (IC10) concentrations of Itra.

Results: Pathway analysis on the top hits from both the screens showed a significant involvement of lysosomal compartments, and in particular dynamics between trans Golgi network and late endosomes/lysosomes, pathways that are affected by the autophagy inhibitor Chloroquine (CQ). We subsequently demonstrated that the combination of Itra and CQ had a synergistic effect in many ovarian cancer cell lines, even in those resistant to Itra. Further, genetic and pharmacological manipulation of autophagy indicated that upstream inhibition of autophagy is not a key mediator of the Itra/CQ mechanism of action. However, combination of Itra with other lysosomotropic agents (Concanamycin A, Bafilomycin A and Tamoxifen) displayed overlapping activity with Itra/CQ, supporting the lysosomal involvement in sensitizing cells to Itra resulted from the CRISPR screens. Analysis of lysosomal pattern and function showed a combined effect of Itra and CQ in targeting lysosomes and neutralizing their activity.

Conclusion: We identified two FDA approved drugs – CQ and Tamoxifen - that can be used in combination with Itra and exert a potent anti-tumor effect in ovarian cancer by affecting lyosomal function and suggesting a likely dependency of these cells on lysosomal biology. Further studies are in progress.

Citation Format: Stefano Marastoni, Aleksandra Pesic, Sree Narayanan Nair, Zhu Juan Li, Ali Madani, Benjamin Haibe-Kains, Bradly G. Wouters, Anthony Joshua. Targeting lysosomal homeostasis in ovarian cancer through drug repurposing [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 3401.
Abstract 3427: Finding critical cancer driving and cancer suppressing genes using functional genomics screening in vivo
Authors: Herold Marco Josef , Mizutani Shinsuke , Deng Yexuan , Janic Ana , Kueh Andrew , Pal Martin , Wilcox Stephen , Tai Lin , Kelly Gemma L. , Strasser Andreas
Published: September 2020

P53 is one of the most potent tumour suppressors and its activity is also required for cancer cells to respond to diverse chemotherapeutic drugs. Focused functional genetic screens in haematopoietic cells in vivo revealed DNA repair to be one of the most critical downstream pathways for p53's tumour suppressive function. Recently, we extended our search for novel tumour suppressor genes acting potentially outside the p53 tumour suppressor pathway. To this end, we performed whole genome CRISPR/Cas9 knockout screening in vivo, using the Eµ-Myc transgenic mouse model. This identified previously unknown genes and hence pathways, which upon deletion/mutation lead to the acceleration of tumour onset. Amongst these novel tumour suppressor genes, we identified the GATOR1 members DEPDC5 and NPRL3, which are known inhibitors of the mTOR pathway. Interestingly, drug mediated inhibition of mTOR activity with rapamycin or Torin1 in GATOR1 deficient Eµ-Myc cells led to efficient killing of these malignant cells, which was in striking contrast to control Eµ-Myc lymphoma cells that were resistant to this treatment. In parallel we conducted CRISPR/Cas9 screens for identifying p53 bound promoter and enhancer elements in haematopoietic cells in vivo. Excitingly, we observed that targeting the NPRL3 promoter led to accelerated tumour onset in the Eµ-Myc transgenic model, suggesting that NPRL3 is under direct transcriptional control of p53. These results indicate that p53 mediated shutdown of the mTOR pathway through upregulation of the GATOR1 member NPRL3 is a critical tumour suppressive function of p53.

Citation Format: Marco Josef Herold, Shinsuke Mizutani, Yexuan Deng, Ana Janic, Andrew Kueh, Martin Pal, Stephen Wilcox, Lin Tai, Gemma L. Kelly, Andreas Strasser. Finding critical cancer driving and cancer suppressing genes using functional genomics screening 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 3427.
Abstract 3428: The role of methylthioadenosine phosphorylase in melanoma
Authors: Hsiao Jennifer Jia-An , Andreu-Perez Pedro , Hejna Miroslav , Kemény Lajos V. , Song Jun , Fisher David E.
Published: September 2020

Methylthioadenosine phosphorylase (MTAP) is an enzyme that is expressed in virtually all normal tissues but lost in many cancers. MTAP deficiency can be due to either deletion of the MTAP gene or methylation of the MTAP promoter. In normal cells, MTAP catalyzes the conversion of methylthioadenosine (MTA), produced during polyamine biosynthesis, to adenine and 5-methylthioribose-1-phosphate, which is subsequently converted to methionine. Although recent genome wide association studies (GWAS) have associated the MTAP locus with melanoma risk, the molecular mechanisms linking MTAP loss to increased tumorigenesis are not yet fully understood. In this study, we hypothesized that loss of MTAP and the resulting accumulation of MTA would have an effect on microphthalmia transcription factor (MITF), the master regulator of melanocytes that has been shown to be an oncogene in melanoma. We present a novel signaling mechanism in which loss of MTAP and subsequent accumulation of MTA induces expression of MITF via inhibition of the phosphodiesterase PDE4D3 by MTA. Inhibition of PKA abolishes the induction of MITF, suggesting that the PKA pathway is hyperactivated when MTAP levels are low. We show that downregulation of MTAP expression leads to increased proliferation of melanoma cells. Using a melanoma xenograft mouse model, we observed that downregulation of MTAP expression leads to increased tumor growth in vivo. These data all point to MTAP as a tumor suppressor in melanoma, and indicate that MTAP loss is an alternative mechanism of dysregulating MITF, a known oncogene in melanoma.

Citation Format: Jennifer Jia-An Hsiao, Pedro Andreu-Perez, Miroslav Hejna, Lajos V. Kemény, Jun Song, David E. Fisher. The role of methylthioadenosine phosphorylase in melanoma [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 3428.
Abstract 3430: The protein phosphatase 4 complex is a tumor suppressor in prostate cancer
Authors: Watson Philip A. , Lawrence Kayla E. , Nadkarni Tejasveeta V. , Ishmael Taslima F. , Lee Eugine , Rivera Aura C. Agudelo , Love Jillian R. , Wongvipat John , Cai Ling , Nazir Abbas , Iaquinta Phillip J. , Chang Kenneth , Liang Yupu , Hannon Greg J. , Sawyers Charles L.
Published: September 2020

Regions of recurring chromosomal deletions have been annotated in clinical prostate cancer (PCa), but for many of them there is an incomplete understanding of their contributions to disease progression. From an in vivo shRNA enrichment screen of the PCa deletome, we identified the serine threonine protein phosphatase 4 (PP4) regulatory subunit 2 (PPP4R2) as a candidate tumor suppressor, which undergoes copy number loss in 16% of primary PCa. Knockdown or CRISPR-Cas9 mediated knockout of PPP4R2 increased the growth rate of PCa xenograft tumors. Along with the catalytic subunit PP4C and an additional regulatory subunit (either PP4R3A or PP4R3B), PP4R2 forms two distinct heterotrimeric phosphatase holoenzymes. PP4R2 was critical for holoenzyme stability, as its deletion resulted in rapid, moderate downregulation of the other PP4 subunit proteins. The increased tumor growth rate and downregulation of PP4C resulting from loss of PP4R2 could be replicated by combined deletion of PPP4R3A and PPP4R3B, or by directly targeting PPP4C itself. Collectively, these findings implicated PP4C as the likely direct mediator of tumor suppression through the PP4 complex. This was confirmed in both PPP4R2 and PPP4R3A/PPP4R3B knockout cells in vivo by restoring PP4C to its normal physiological level through a dox-inducible cDNA, which reversed the accelerated tumor growth rate conferred by the deletion of the regulatory subunits. Phosphatase activity of PP4C was critical, as add-back of a catalytically dead PP4C mutant (D82A) was unable to reverse the growth rate of PPP4C knockout tumors. Interestingly, we found that the androgen receptor (AR), which is the principal therapeutic target for PCa, was co-immunoprecipitated by PP4C and its regulatory subunits. This suggests that AR could be a substrate for PP4C and is potentially present in a hyperphosphorylated and hyperactivated state in PCa that have decreased PP4 phosphatase activity. Our findings highlight a previously unappreciated tumor suppressor role for the PP4 complex in PCa, with the regulatory subunit PP4R2 providing a key function in stabilizing PP4C and thereby preserving its phosphatase activity.

Citation Format: Philip A. Watson, Kayla E. Lawrence, Tejasveeta V. Nadkarni, Taslima F. Ishmael, Eugine Lee, Aura C. Agudelo Rivera, Jillian R. Love, John Wongvipat, Ling Cai, Abbas Nazir, Phillip J. Iaquinta, Kenneth Chang, Yupu Liang, Greg J. Hannon, Charles L. Sawyers. The protein phosphatase 4 complex is a tumor suppressor in prostate cancer [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 3430.