bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1456: First successful engraftment of human liver cancer cell line in highly robust immunocompromised porcine model to test the tumor ablation efficacy by histotripsy
Authors: Paul Tamalika , Imran Khan M. , Gannon Jessica , Trusiano Brie , Eden Kristin , Ivester Hannah , Mott Madeline , Powar Manali , Edwards Michael , Byron Christopher , Clark-Deener Sherrie , Lee Kiho , Vlaisavljevich Eli , Allen Irving Coy
Published: March 2024

Liver tumor, commonly called Hepatocellular carcinoma(HCC), is the most common type of primary liver malignancy. It is the fourth leading cause of cancer-related deaths. Late diagnosis, location of the tumor, tumor burden, and metastases makes liver tumor very challenging to treat by liver transplantation, surgical procedures, and other ablation techniques like cryoablation and thermal ablation. Also, the absence of a preclinical animal model makes it challenging to develop and explore the feasibility of treatment modalities. Histotripsy is a non-invasive, non-ionizing, non-thermal, image-guided focused ultrasound ablation treatment method that uses high-pressure pulses to create acoustic cavitation, a “bubble cloud,” at the target. The bubble cloud expands and collapses, which ablates the tissue into an acellular homogenate. Pigs are ideal animal models as they closely resemble human anatomy and are significant in improving the translation to human patients. This study aimed to establish the feasibility of successfully ablating liver tumors by histotripsy using an immunocompromised porcine model to generate HepG2 human liver cancer cell line tumors in the liver of the immunocompromised pigs. The orthotopic porcine model was established using RAG2/IL2RG double knockout immunocompromised pigs. HepG2 cells were injected orthotopically into the liver of immunocompromised pigs. Three weeks after the injections, CT images and necropsy indicated successful engraftment and growth of liver tumors in the pigs. The ultrasound images and post-histotripsy treatment histology images showed confirmed ablation zones in the liver. Therefore, in conclusion, our preliminary results in the study could demonstrate, for the first time, a highly robust model of human liver cancer in a large animal model. Soon, we plan to conduct more such studies and explore all the possible utilities of these immunocompromised porcine models to develop therapeutic strategies to increase the efficacy of liver tumor ablation by histotripsy.

Citation Format: Tamalika Paul, Khan M. Imran, Jessica Gannon, Brie Trusiano, Kristin Eden, Hannah Ivester, Madeline Mott, Manali Powar, Michael Edwards, Christopher Byron, Sherrie Clark-Deener, Kiho Lee, Eli Vlaisavljevich, Irving Coy Allen. First successful engraftment of human liver cancer cell line in highly robust immunocompromised porcine model to test the tumor ablation efficacy by histotripsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1456.
Abstract 1485: Hallmarks of APOBEC3 mutagenesis in normal, pre-leukemic, and myeloproliferative neoplasm cell populations
Authors: Isquith Jane Marie , Whisenant Thomas , Pham Jessica , Alexandrov Ludmil , Jamieson Catriona
Published: March 2024

Changes in gene expression and subsequent editing patterns of APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like) cytosine deaminases have been shown to be present in solid tumor cancer evolution, however, the context specificity and mechanisms by which APOBEC3 enzymes promote cancer initiation and progression require further elucidation, especially in the hematopoietic niche. Building on advanced sequencing data of healthy, pre-leukemic, and myeloproliferative neoplasm patient samples, we seek to uncover normal and malignant patterns of APOBEC3C overexpression in hematopoietic cells. Overexpression of lentiviral APOBEC3C and an editase-mutated APOBEC3C in normal cord blood, healthy bone marrow, and MPN patient hematopoietic stem/progenitor cells (HSPCs) allows us to study the effects of deaminase dysregulation in hematopoietic cells. In addition to APOBEC3, we are exploring the upregulation of adenosine deaminase acting on RNA1 (ADAR1), as we have previously shown that these two innate immune deaminases are synchronously upregulated in the high-risk myelofibrosis (MF) stem cell population compared to normal aged bone marrow. These overexpression studies show novel differential gene expression changes, RNA hyper-editing sites, and DNA mutation signatures induced by APOBEC3 mutagenesis, which can be cross-referenced to gene expression and mutation signatures seen in hematopoietic malignancies and solid tumor cancers. In addition to mutation patterns, the regulation of LINE-1 elements has been shown to be another important function of APOBEC3 enzymes. In these studies, we examine the effects of APOBEC3 overexpression on repetitive element expression in hematopoietic cell populations and have found that there are strong cell type and context patterns across the APOBEC3 family and between cell types within the hematopoietic system. We will use this comprehensive study of APOBEC3 deregulation to uncover predictive biomarkers of malignant transformation.

Citation Format: Jane Marie Isquith, Thomas Whisenant, Jessica Pham, Ludmil Alexandrov, Catriona Jamieson. Hallmarks of APOBEC3 mutagenesis in normal, pre-leukemic, and myeloproliferative neoplasm cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1485.
Abstract 1490: Elucidating the effect of glutamine metabolism in breast to bone metastasis
Authors: Karno Breelyn A. , Hwang Yoonha , Johnson Rachelle , Chen Jin , Edwards Deanna
Published: March 2024

Bone-metastatic lesions will develop in approximately 65-75% of patients with metastatic breast cancer and are associated with high morbidity and mortality. Despite our limited understanding of how breast-to-bone metastases form, the recent emergence of metabolic adaptation as a critical factor in metastasis is a promising development. Breast cancer cells are particularly reliant on glutamine, a key amino acid that is metabolized by the enzyme glutaminase (GLS) to facilitate many downstream metabolic processes. Bone-tropic breast cancer cell lines significantly enhance their glutamine uptake, suggesting that glutamine metabolism may be a significant factor in bone metastasis. In addition to tumor cells, glutamine metabolism is critical for osteoblasts, important bone-resident cells that contribute to the “vicious cycle” of bone destruction during metastatic outgrowth in the bone niche. In this study, we sought to evaluate the role of glutamine metabolism in breast cancer bone metastasis by examining the effect of GLS function in metastasizing tumor cells and bone-native osteoblasts. Using an intracardiac inoculation model of bone metastasis, we found that breast cancer cells lacking GLS (GLS KO) failed to effectively form bone metastatic tumors. We also observed that GLS KO bone tumors show a reduction in osteolysis using a direct intratibial injection model. Mechanistically, we have found that GLS KO breast cancer cells develop a “senescence-like” phenotype that may limit metastatic outgrowth within the bone. To investigate the importance of glutamine metabolism in the bone niche, we developed a conditional genetic mouse model to delete GLS in osterix-expressing in osteoblast progenitors and mature osteoblasts (GLSOBKO). Remarkably, we observed reduced bone destruction in GLSOBKO mice after intratibial injection of syngeneic breast cancer cells. We also report a reduction in the number of osteoclasts at the bone/tumor interface in these mice, suggesting that loss of osteoblast GLS could temporarily halt the “vicious cycle” of bone metastasis. Together, this work suggests that glutamine metabolism may promote breast-to-bone metastasis via both intrinsic and extrinsic mechanisms. These findings raise the possibility of using pre-existing cancer therapies targeting glutamine metabolism to ameliorate bone metastatic burden in breast cancer.

Citation Format: Breelyn A. Karno, Yoonha Hwang, Rachelle Johnson, Jin Chen, Deanna Edwards. Elucidating the effect of glutamine metabolism in breast to bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1490.
Abstract 1504: Tumor microenvironment-mediated regulation of early dissemination and colonization in breast cancer via BAZ2A-containing remodeling complexes
Authors: Kokal-Ribaudo Miriam , Jahnen Nora , Sameri Saba , Ferrazzi Fulvia , Liguori Renato , Klein Christoph A. , Längst Gernot , Hosseini Hedayatollah
Published: March 2024

The molecular mechanisms governing invasion, dissemination, extravasation, and early metastatic colonization remain incompletely understood. In the BALB-neuT mouse model, we found that most metastases originate from disseminated cancer cells (DCCs) that left the primary site early in tumorigenesis, i.e. when lesions are still small. Consequently, we investigated whether cellular density and the tumor microenvironment synergize to generate the plasticity needed during metastasis.

RNA-sequencing analyses of murine breast cancer cells in low cellular density (LD) and high cellular density (HD) conditions revealed distinct profiles of differentially expressed genes. Notably, we identified the enrichment of ribosomal RNA (rRNA) and chromatin remodeling pathways in LD conditions. Interestingly, once these cells were treated with Wnt4 and Rankl, factors secreted by the tumor microenvironment, the expression profile of genes involved in proliferation, migration and stemness as well as epigenetic-related factors changed drastically. Specifically, cells in LD conditions treated with Wnt4 and Rankl displayed induced migration and stemness phenotypes, whereas under HD conditions, these signals induced proliferation. Among the chromatin-remodeling pathways, we found that BAZ2A along with the rRNA regulating network were primarily upregulated in migratory cancer cells. Immunostaining of primary and metastatic lesions in murine and human breast cancer samples showed an upregulation of BAZ2A in early/microlesions compared to more advanced and proliferative macroscopic lesions.

Taken together, these results indicate that cell density, tumor microenvironmental factors and BAZ2A-containing chromatin regulating complexes play pivotal roles in regulating cancer cell plasticity in breast cancer. Additional sequencing and functional analyses will be performed to dissect the specific components of this mechanism and evaluate their impact on the in vivo colonization of breast cancer models.

Citation Format: Miriam Kokal-Ribaudo, Nora Jahnen, Saba Sameri, Fulvia Ferrazzi, Renato Liguori, Christoph A. Klein, Gernot Längst, Hedayatollah Hosseini. Tumor microenvironment-mediated regulation of early dissemination and colonization in breast cancer via BAZ2A-containing remodeling complexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1504.
Abstract 1514: A novel workflow to assess the T-cell and patient-derived organoid interaction
Authors: Tong Zhisong , Lim Angeline , Sirenko Oksana
Published: March 2024

Immunotherapy is increasingly popular as a type of cancer treatment. These therapies include the use of Chimeric Antigen Receptor-engineered T-cells (CAR T-cells), tumor-infiltrating lymphocytes (TIL), and other genetically modified T-cells to specifically target the cancer cells. Although much success has been achieved with immunotherapy for treatment of blood cancers, its efficacy remains limited in solid tumors. One of the reasons for the low success rate is attributed to the solid tumor microenvironment (TME) where suppressive cytokines limit the tumor killing ability of T-cells. Thus, understanding the role the TME plays in T-cell responses is essential for the development of effective cancer therapies. The benefits of using 3-dimensional (3D) PDOs lie in the physical and chemical cues present within the TME that cannot be mimicked in traditional 2D monolayer cultures. Studies show that PDOs show similar responses to drugs as original tumors, suggesting the value of using PDOs to improve therapeutic outcomes. Thus, PDOs can provide more relevant physiological and pathological cancer models that recapitulate the basic features of primary tumors and is more suited for assessing the effectiveness of T-cell killing than 2D cell models. Despite the benefits associated with the use of PDOs, there are significant barriers to widespread adoption of PDOs in drug discovery. Organoid production is a costly and highly labor-intensive process. Moreover, organoid culture is a skilled manual process, and thus there can be significant variability between operators. To address the challenges associated with the use of PDOs in large scale applications, a semi-automated bioprocess has been developed for the large-scale expansion of assay ready organoids. Here, we developed a method to assess the effectiveness of T-cell invasion in solid tumors using patient derive organoids (PDOs). Using bioreactor expanded patient-derived colorectal cancer organoids (CRCs), activated PBMCs (human peripheral blood mononuclear cells) stained with CellTracker were added to CRCs (stained with MitoTracker) in a 96well microtiter plate and monitored on every 4 hours for 3 days using high content imager. To quantify T-cell invasion, we developed an image analysis method to measure the distance of each T-cell to the nearest organoid (interaction distance). We find that stimulated T-cell resulted in smaller interaction distance than non-stimulated T-cells. The results demonstrate the utility of the bioreactor-expanded organoids in large scale T-cell based screens.

Citation Format: Zhisong Tong, Angeline Lim, Oksana Sirenko. A novel workflow to assess the T-cell and patient-derived organoid interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1514.
Abstract 1539: A comprehensive analytical validation framework for antibodies used in the CellScape multiplex immunofluorescence assay
Authors: Jackson Charles E. , Christians Arne , Boog Jannik , Ingalls Matthew H. , Meshik Xenia , Northcutt Adam , Cashion Kayla E. , Schwarz J Spencer , Braubach Oliver
Published: March 2024

Background: Multiplex immunofluorescence (mIF) is a powerful tool that enables deep phenotyping of cells within the tumor microenvironment (TME). Careful optimization of multiplex antibody panels for specificity, sensitivity and reproducibility is crucial to achieving robust data and is a precondition for assay repeatability and standardization. We describe our validation procedure for VistaPlex™ multiplex assay panels.

Methods: Experiments were performed using the CellScape™ platform for precise spatial multiplexing. The platform enables automated cyclic mIF for readily available fluorophore-conjugated antibodies compatible with fresh frozen or FFPE tissues. The use of high dynamic range imaging further facilitates quantitative phenotyping, while excellent imaging resolution provides single-cell discrimination with high accuracy. To design a ready-to-use panel for immune cell phenotyping, individual antibodies were first screened for specificity and then optimized for labeling sensitivity. Signal-to-noise, inter-assay reproducibility, and intra-assay precision were evaluated across serial sections in a multi-stage testing matrix to validate each antibody in isolation and assembled into a panel. Resulting from this effort, we introduce a 15-plex ready-to-use VistaPlex immune profiling panel for human FFPE tissues.

Results: Image analysis of signal-to-noise ratios demonstrated robust performance of multiplexed antibodies across multiple tissue sections and tissue types. Measurements of both signal and quantitative immune phenotyping across technical replicates showed a high degree of intra-assay precision and inter-assay reproducibility. The 15-plex panel was validated on human tonsil, lung, breast, and colon tissues.

Conclusions: The CellScape platform enables simultaneous detection of multiple protein biomarkers on a single tissue section for deep immune cell profiling in the TME. Rigorously validated VistaPlex panels further streamline this spatial biology workflow and will aid in the generation of consistent and reliable results for end users.

Citation Format: Charles E. Jackson, Arne Christians, Jannik Boog, Matthew H. Ingalls, Xenia Meshik, Adam Northcutt, Kayla E. Cashion, J Spencer Schwarz, Oliver Braubach. A comprehensive analytical validation framework for antibodies used in the CellScape multiplex immunofluorescence assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1539.
Abstract 1542: Epithelial cell adhesion molecule induces canonical Wnt pathway to promote stemness in colorectal cancer
Authors: Wu Han-Chung , Panda Sushree Shankar , Chen Kai-Chi , Lee Chi-Chiu , Hsieh Hsing-Pang , Su Yi-Jen
Published: March 2024

Epithelial cell adhesion molecule (EpCAM) is a pleiotropic type-1 transmembrane glycoprotein and known cancer stem cell marker, yet its tumorigenic mechanisms remain elusive. Cancer stem cells (CSC) have been implicated in cancer progression and recurrence, but targeting them with therapeutics is a major challenge. It is known that EpCAM is highly expressed in CSCs of different cancer types. Here, we studied the role of EpCAM in colorectal cancer (CRC) stemness. First, we show that the extracellular domain of EpCAM (EpEX) functions as a ligand for Wnt receptors, frizzled6/7 and LRP5/6, while the intracellular domain (EpICD) upregulates transcription of these receptors. Further, EpEX-induced Wnt signaling activates TACE and γ-secretase enzymes to augment shedding of EpEX and EpICD, establishing a positive feedback loop. Importantly, we show that the EpCAM-neutralizing antibody, EpAb2-6, targets the Wnt pathway to attenuate stemness characteristics in colorectal cancer (CRC). Our EpCAM-neutralizing antibody (EpAb2-6) and a porcupine inhibitor (LGK974) each partially attenuated cancer stemness, while their combination abolished stemness-related endpoints, induced apoptosis in vitro and markedly diminished tumor progression in animal models of human CRC. From these findings, we conclude that EpCAM stimulates Wnt signaling to promote cancer stemness, and the combination of EpAb2-6 and porcupine inhibitors may represent an effective CRC treatment, including for KRAS-mutant cancers. Our findings reveal an opportunity to combat drug resistance in tumor stem cells. Thus, the mechanistic insights gained from our study may be useful to improve existing treatments or to develop novel anticancer therapeutics.

Citation Format: Han-Chung Wu, Sushree Shankar Panda, Kai-Chi Chen, Chi-Chiu Lee, Hsing-Pang Hsieh, Yi-Jen Su. Epithelial cell adhesion molecule induces canonical Wnt pathway to promote stemness in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1542.
Abstract 1552: Tumorigenesis of basal muscle invasive bladder cancer was mediated by PTEN protein degradation resulting from SNHG1 upregulation
Authors: Huang Chuanshu , Li Tengda , Jin Honglei
Published: March 2024

hosphatase and tensin homolog deleted on chromosome ten (PTEN) serves as a powerful tumor suppressor, and has been found to be downregulated in human bladder cancer (BC) tissues. Despite this observation, the mechanisms contributing to PTEN’s downregulation have remained elusive. In this study, we discovered that mice exposed to N-butyl-N-(4-hydroxybu-tyl)nitrosamine (BBN), a specific bladder chemical carcinogen, exhibited primary basal muscle invasive BC (BMIBC) accompanied by a pronounced reduction in PTEN protein expression in vivo. Utilizing a lncRNA deep sequencing high-throughput platform, along with gain- and loss-of-function analyses, we identified small nucleolar RNA host gene 1 (SNHG1) as a critical lncRNA that might drive the formation of primary BMIBCs in BBN-treated mice. Cell culture results further demonstrated that BBN exposure significantly induced SNHG1 in normal human bladder urothelial cell UROtsa. Notably, the ectopic expression of SNHG1 alone was sufficient to induce malignant transformation in human urothelial cells, while SNHG1 knockdown effectively inhibited anchorage-independent growth of human BMIBCs. Our detailed investigation revealed that SNHG1 overexpression led to PTEN protein degradation through its direct interaction with HUR. This interaction reduced HUR binding to ubiquitin-specific peptidase 8 (USP8) mRNA, causing degradation of USP8 mRNA and a subsequent decrease in USP8 protein expression. The downregulation of USP8, in turn, increased PTEN poly-ubiquitination and degradation, culminating in cell malignant transformation and BMIBC anchorage-independent growth. In vivo studies confirmed the downregulation of PTEN and USP8, as well as their positive correlations in both BBN-treated mouse bladder urothelium and tumor tissues of bladder cancer in nude mice.Our findings, for the first time, demonstrate that overexpressed SNHG1 competes with USP8 for binding to HUR. This competition attenuates USP8 mRNA stability and protein expression, leading to PTEN protein degradation, consequently, this process drives urothelial cell malignant transformation and fosters BMIBC growth and primary BMIBC formation.

Citation Format: Chuanshu Huang, Tengda Li, Honglei Jin. Tumorigenesis of basal muscle invasive bladder cancer was mediated by PTEN protein degradation resulting from SNHG1 upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1552.
Abstract 1564: NEU4-mediated desialylation ignites the oncogenic receptors for the dissemination of ovarian carcinoma
Authors: Shi Jie , Zhou Rui , Wang Shuo , Liu Yuxin , Tian Baorui , Liu Yanhua , Chen YaNan , Hu Taoyu , Mu Yuhao , Wang Shufan , Shao Xintao , Yan Jie , Qu Pengpeng , Ding Wei , Yang Shuang , Shi Yi , Li Jia , Wang Longlong
Published: March 2024

Background: Glycosylation profoundly influences the interactions between cancer cells and microenvironmental stromal cells during the peritoneal disseminated metastasis of ovarian carcinoma (OC), which is the major cause of cancer-related death. Although the characteristic cancer glycoconjugates are widely used as biomarkers for cancer diagnosis, our knowledge about cancer glycome remains quite fragmented due to the technique limitations in analyzing glycan chains with tremendous structural and functional heterogeneity.

Method: Given the dysregulated cancer glycome is defined by the altered glycosylation machinery, here we performed a systematic loss-of-function screen on 498 genes involved in glycosylation for key regulators of OC dissemination. We identified neuraminidase 4 (NEU4), an enzyme capable of hydrolyzing terminal sialic acid from glycoconjugates, as a vital peritoneal dissemination-promoting modifier of OC glycome. What’s more, we also identified membrane proteins whose sialylation was able to be modified by NEU4 in OC cells by pull down and mass spectrum to investigate the functional significance of NEU4-mediated desialylation.

Results: In human patients with high-grade serous OC (HGSOC), increased NEU4 was detected in the disseminated OC cells when compared with that in the primary tumor cells, which significantly correlated with the worse survival. Among three alternative splice-generated isoforms of human NEU4, we revealed that only the plasma membrane-localized NEU4 isoform 2 (NEU4-iso2) and intracellular isoform 3 promoted the peritoneal dissemination of OC by enhancing the cell motility and epithelial-mesenchymal transition. We also identified NEU4-iso2-regulated cell surface glycoproteome and found that NEU4-iso2 desialylated the epithelial growth factor receptor (EGFR), in particular at N196 residue, for the hyperactivation of EGFR and its downstream tumor-promoting signaling cascades.

Conclusion: Our results provide new insights into how the OC glycome is dysregulated by NEU4 during OC progression and reveals a functionally important glycosite on EGFR for its abnormal activation in cancer.

Citation Format: Jie Shi, Rui Zhou, Shuo Wang, Yuxin Liu, Baorui Tian, Yanhua Liu, YaNan Chen, Taoyu Hu, Yuhao Mu, Shufan Wang, Xintao Shao, Jie Yan, Pengpeng Qu, Wei Ding, Shuang Yang, Yi Shi, Jia Li, Longlong Wang. NEU4-mediated desialylation ignites the oncogenic receptors for the dissemination of ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1564.
Abstract 1586: Unraveling the importance of pancreatic cancer extracellular signaling to endothelial cells within the tumor microenvironment
Authors: Finan Jennifer M. , Guo Yifei , Bartlett Alexandra Q. , MacPherson Kevin , Calvert Valerie , Hedberg Madeline D. , Petricoin Emanuel , Sears Rosalie , Brody Jonathan R.
Published: March 2024

In many solid tumors, including pancreatic ductal adenocarcinoma (PDAC), the secretion of extracellular vesicles (EVs) has been shown to support tumor progression, chemotherapeutic resistance, and metastasis. Importantly, PDAC tumors are composed of up to 80% non-tumor cells and an extracellular matrix, yet no studies have been performed to determine which cells within PDAC tumors internalized PDAC EVs. Further, the RNA-binding protein HuR plays an important role in PDAC cells, supporting the stability and translation of transcripts that aid tumor cell stress responses. Our lab has recently found that EVs harbor distinct mRNA and phosphorylated protein cargoes from donor cells. Further, HuR knockout (KO) cells produce EVs with significantly different mRNAs and phosphorylated proteins that EVs isolated from HuR wildtype (WT) cells. Specifically, proteins relating to endothelial function and angiogenesis were significantly differentially phosphorylated. Considering the functional impact of EV cargoes on recipient cells, our study aims to identify the specific cell types in the tumor microenvironment that import PDAC EVs and investigate the role of PDAC cell HuR in this signaling process. We have generated human PDAC and mouse KrasG12D Trp53R172H driven (KPC) PDAC cell lines to express an EV reporter, PalmGRET. Utilizing these cells lines in an immunocompetent mouse model of PDAC, we found that endothelial cells are major importers of PDAC EVs. We next performed bulk RNA-sequencing on endothelial cells sorted from these tumors that have (GFP+) versus have not (GFP-) imported PDAC EVs to assess the functional impact of PDAC EVs. Subsequent studies will focus on delineating the importance of this signaling axis by ablating EV secretion in addition to assessing endothelial cell function when treated with EVs from HuR WT vs. KO cells. Collectively, our study sheds light on the preferential uptake of PDAC EVs by endothelial cells and highlights the potential role of tumor cell-intrinsic HuR in this signaling process. These findings expand our understanding of the interactions between PDAC and the tumor microenvironment, potentially paving the way for targeted therapeutic interventions in this devastating disease.

Citation Format: Jennifer M. Finan, Yifei Guo, Alexandra Q. Bartlett, Kevin MacPherson, Valerie Calvert, Madeline D. Hedberg, Emanuel Petricoin, Rosalie Sears, Jonathan R. Brody. Unraveling the importance of pancreatic cancer extracellular signaling to endothelial cells within the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1586.
Abstract 159: LYVE-1-expressing macrophages modulate the hyaluronan-containing extracellular matrix in the mammary stroma and contribute to mammary tumor growth
Authors: Elfstrum Alexis K. , Rumahorbo Annisa H. , McCluskey Braedan M. , Schwertfeger Kathryn L.
Published: March 2024

Macrophages represent a heterogeneous myeloid population with diverse functions in normal tissues and tumors. While macrophages expressing the cell surface marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) have been identified in stromal regions of the normal mammary gland and in the peri-tumoral stroma, their functions within these regions are not well-understood. Using a genetic LYVE-1+ macrophage deletion mouse model, we demonstrate that loss of LYVE-1+ macrophages is associated with altered extracellular matrix remodeling in the normal mammary gland and reduced mammary tumor growth in vivo. In further studies focused on investigating the functions of LYVE-1+ macrophages in the tumor microenvironment, we demonstrate that LYVE-1 expression correlates with an increased ability of macrophages to bind, internalize, and degrade hyaluronan. Consistent with this, we show that deletion of LYVE-1+ macrophages correlates with increased hyaluronan accumulation in both the normal mammary gland and in mammary tumors. Analysis of single cell RNA sequencing of macrophages isolated from these tumors reveals that deletion of LYVE-1+ macrophages in tumors drives a shift in the majority of the remaining macrophages towards a pro-inflammatory phenotype, as well as an increase in CD8+ T cell infiltration. Together, these findings demonstrate that LYVE-1+ macrophages represent a tumor-promoting anti-inflammatory subset of macrophages that contributes to hyaluronan remodeling in the tumor microenvironment.

Citation Format: Alexis K. Elfstrum, Annisa H. Rumahorbo, Braedan M. McCluskey, Kathryn L. Schwertfeger. LYVE-1-expressing macrophages modulate the hyaluronan-containing extracellular matrix in the mammary stroma and contribute to mammary tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 159.
Abstract 1599: Metabolism as a key regulator of macrophage phenotype in pancreatic cancer
Authors: Anaraki Cecily , Halbrook Christopher
Published: March 2024

Pancreatic Ductal Adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related deaths in the US, with a five-year survival rate of 12%. Barriers to treatment include the highly fibrotic tumor microenvironment (TME), the high interstitial pressure which acts to collapse the blood vessels, and the influx of immunosuppressive immune cells. Within this immune milieu, we find that the most abundant are tumor-associated macrophages (TAMs). These TAMs display a phenotype similar to anti-inflammatory (M2-like) macrophages. Despite TAMs exerting an immunosuppressive function within the TME, there is not a complete overlap with the characteristics associated with M2 macrophages. Interestingly, TAMs still display many markers associated with the pro-inflammatory (M1-like) macrophages. This dichotomy suggests there may be a level of plasticity within the macrophage compartment that can be exploited to lead to better tumor control. Previous studies have shown a connection between arginine metabolism and macrophage effector function along the M1/M2 axis. In macrophages, arginine can be broken down in two ways: via iNOS into nitric oxide, which aids in effector function, or via Arginase 1 (Arg1) into ornithine and later proline, which is an essential component of the extracellular matrix (ECM). This study seeks to elucidate how metabolic perturbations influence macrophage function and phenotype. Here we observed that changes in oxygen availability and glucose abundance shifted macrophage expression of iNOS and Arg1. In vivo we found that conditionally knocking out Arg1 in the myeloid compartment leads to lower fibrosis when inducing inflammation in the pancreas. Furthermore, macrophages polarized with PDAC cancer-conditioned media display a sharp increase in Arg1 expression as compared to unpolarized macrophages, as well as increased resistance to both glycolytic inhibitors and mitochondrial inhibitors. This preliminary data demonstrates an important link between macrophage metabolism and function in the context of pancreatic disease.

Citation Format: Cecily Anaraki, Christopher Halbrook. Metabolism as a key regulator of macrophage phenotype in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1599.
Abstract 1669: KRAS G12X mutants display differential preferences in downstream effector binding
Authors: Ash Leonard J. , Bourdain Ottavia , Lam Dennis , Pallas Shlomo S. , Wolfe Andrew L.
Published: March 2024

KRAS is among the most frequently mutated oncogenic driver proteins in human cancers including lung, colorectal, and pancreatic cancers. Activating KRAS mutants cause increased cell proliferation through direct binding and overactivation of effector proteins. Effector activation preference is driver mutation specific, as KRAS G12D and G12C purportedly bind both RAF and PI3K, whereas G12R has been reported to bind RAF but not PI3K. The mechanisms of effector binding of G12S action are under-investigated. Furthermore, most KRAS mutations remain clinically untreatable, as clinically approved KRAS inhibitors (KRASi) are currently limited to specifically targeting G12C. While experimental non-G12C inhibitors are being developed, resistance mechanisms to non-G12C mutations—including G12D/R/S/V—remain relatively understudied.

We set out to characterize the binding efficiency of KRAS to RAF1 and the PI3K subunit p110α, and to understand how isoforms differ in inhibitor resistance strategies. Our findings suggest that significant variances in KRAS mutation-specific effector binding preference exist and are cell line dependent. We performed proximity ligation based immunofluorescence assays (PLA) on an isogenic panel of human colorectal SW48 KRAS cells with G12C/D/R/S/V or wildtype. SW48 cells with G12S significantly prefer the KRAS-RAF1 mode of binding over otherwise isogenic WT cells, and G12V homologs prefer PI3K. Our findings suggest that in this model, G12R cells have no specific preference of one binding mode over the other. We hypothesize that G12 chemistry drives effector binding preference and that G12X inhibitor resistant cell cultures will demonstrate variant mechanisms of resistance in line with their preferred binding partner. Ongoing studies will further characterize understudied RAS isoform biology and evaluate the links between isoform chemistry and mechanisms of inhibitor resistance. These studies will expand our mechanistic knowledge of the interplay between G12X chemistry and adaptive resistance in human cancer.

Citation Format: Leonard J. Ash, Ottavia Bourdain, Dennis Lam, Shlomo S. Pallas, Andrew L. Wolfe. KRAS G12X mutants display differential preferences in downstream effector binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1669.
Abstract 1674: Inhibition of oncogenic nras signaling in acute myeloid leukemia cell lines through directed transcriptome alterations
Authors: Roberts Michael , Kovatsis Sophia , Harper Amelia , Cravinho Megan , Mangru Kishan , Bahadur Sher , Hesse Keagan , Meta Katy , Nguyen Cuong
Published: March 2024

Oncogenic mutations in genes involved in proliferative signaling are hallmarks of malignant transformation. The Ras family of signal transducing proteins are involved in the activation of several proliferative pathways and therefore are found to be oncogenically mutated in a variety of cancer types. The NRAS-directed pathways resulting in Erk1/2 activation are dysregulated in a significant percentage of acute myeloid leukemia (AML), metastatic melanoma, Hodgkin lymphoma, and thyroid cancers. The consequence of oncogenic NRAS signaling is the establishment of a transcriptome commanding constitutive proliferation and survival. It has long been known that phorbol ester treatment of AML cell lines, resulting in activation of protein kinase C (PKC), causes phenotypic changes that include cell cycle arrest, myeloid differentiation, and apoptosis. The paradox is that numerous studies identify PKC as an activator of RAS/ERK signaling. Here we show that phorbol ester treatment of NRAS-driven AML cell lines results in the transcriptional upregulation of genes encoding negative regulators of RAS signaling including DUSP1,2,4,5,6, SPRY2, SPRED1, RASA1, PEA15, RPS6KA1, and RASSF5. Additionally, phorbol ester treatment eventually results to ERK1/2 inactivation through dephosphorylation. Several of these negative regulators, when overexpressed in AML cell lines, dampen proliferation, and induce senescence. We propose that the induction of RAS negative regulators alters the AML transcriptome to a non-proliferative state in part through the transcriptional regulation of genes such as CDKN1A, CDKN2B, ZFP36L1, and MYC. As gene therapy strategies develop, designed to provide negative regulators of proliferative signaling pathways that are repressed in the malignant cell, we imagine the RAS/ERK pathway as a target for transcriptome reprogramming.

Citation Format: Michael Roberts, Sophia Kovatsis, Amelia Harper, Megan Cravinho, Kishan Mangru, Sher Bahadur, Keagan Hesse, Katy Meta, Cuong Nguyen. Inhibition of oncogenic nras signaling in acute myeloid leukemia cell lines through directed transcriptome alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1674.
Abstract 1675: Investigating the role of SRC-family kinases in MPNST tumorigenesis
Authors: Zereg Elamine , Voisin Laure , Grenier Karl , Courcelles Mathieu , Jung Sungmi , Thibault Pierre , Meloche Sylvain
Published: March 2024

Purpose: Malignant peripheral nerve sheath tumor (MPNST), an aggressive soft-tissue sarcoma, is the leading cause of mortality in patients with neurofibromatosis type 1. Over the past two decades, significant advancement has been made in understanding the pathogenesis of MPNST. Still, this progress has not improved overall survival of patients. Therefore, ongoing efforts to identify alterations contributing to the transformation, progression, and metastasis of MPNST are essential for pinpointing targetable oncogenic signaling pathways for this cancer. Previous studies have revealed an increase in YAP/TAZ activity through both LATS1/2-dependent and LATS1/2-independent mechanisms, suggesting that YAP/TAZ hyperactivity may act as a convergence point for MPNST pathogenesis. YES and SRC, two members of the SRC-family kinases (SFKs), were shown to phosphorylate LATS1/2 and YAP/TAZ, and stimulate the transcriptional activity of YAP/TAZ by promoting their nuclear accumulation in hepatocellular carcinoma and colorectal cancer cells. Thus, we have investigated whether YES and SRC are involved in the pathogenesis of MPNSTs.

Experimental Design: To assess whether the SFK-YAP/TAZ signaling axis is implicated in the development of MPNST, we have used genetic and pharmacological approaches to investigate the roles of YES and SRC in the proliferation of a subset of human MPNST cell lines, and in the growth of xenografted MPNST tumors in ectopic and orthotopic mouse models. We also performed a phosphoproteome and a transcriptome analysis of YES and SRC-depleted MPNST cells to assess their oncogenic signaling network. Lastly, we evaluated the clinical significance of phospho-SFK and YAP in human MPNST TMAs.

Results: Our findings reveal that YES and SRC activity play an essential and redundant role in sustaining the proliferation of human MPNST cell lines. Further, the simultaneous depletion of both kinases impedes tumor growth in xenograft MPNST mouse models. Transcriptomic analyses identified YAP and TAZ as potential effectors of SFKs, as well as other family of transcription factors. Finally, TMA data suggested a correlation between the expressions of YAP, YAP/TAZ, and phospho-SFK and the progression of MPNST.

Citation Format: Elamine Zereg, Laure Voisin, Karl Grenier, Mathieu Courcelles, Sungmi Jung, Pierre Thibault, Sylvain Meloche. Investigating the role of SRC-family kinases in MPNST tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1675.
Abstract 1678: Docetaxel induces Rac1 and Cdc42 Rho GTPase activation in prostate cancer cells
Authors: González Andrea Paola López , González Jessica Colón , Fuentes María C. Santa María , Dharmawardhane Surangani , Osterman Carlos J. Diaz
Published: March 2024

Prostate cancer (PCa) is a major health burden that affects the quality of life of men worldwide. In the United States, PCa is the second leading cause of mortality among cancers that affect men. Treatment of advanced PCa aims to suppress androgen signaling and proliferation in cancer cells using anti-androgens and chemotherapy. However, the development of drug resistance is a major limitation to curing advanced prostate cancer and strategies to block mechanisms of therapy resistance would enhance treatment efficacy and potentially improve patient prognosis. Cdc42 and Rac1, members of the family of Rho GTPases, have been associated with the progression of different types of hormone-associated cancer, such as breast, ovary, and prostate, among others. The current study was designed to evaluate the role of Cdc42 and Rac1 in chemoresistant prostate cancer cells, including under castration-resistant and androgen-independent conditions. We did not observe significant differences in active (Rac-GTP) or total Rac1 levels in chemoresistant compared to parental PC3 or DU145 cells; similar findings were observed for Ccd42. Treatment of drug-naïve cells with a sublethal dose of docetaxel (10uM) induced gradual increases in Rac1-GTP up to 30 minutes in 22rv1, 60 minutes in LNCaP and DU145, and 24 hours in PC3 cells. Cdc42-GTP levels also increased with subacute docetaxel treatment up to 24 hours in 22rv1 and DU145. Chemoresistant 22rv1, PC3, and DU145 exhibited similar sensitivity to Rac1 and Ccd42 inhibitors including MB1-167, MBQ-168, EHop-097, with an IC50 of about 1uM for each compound and line. Ongoing studies are evaluating the role of these Rho-GTPases in 3-dimensional growth and survival.

Citation Format: Andrea Paola López González, Jessica Colón González, María C. Santa María Fuentes, Surangani Dharmawardhane, Carlos J. Diaz Osterman. Docetaxel induces Rac1 and Cdc42 Rho GTPase activation in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1678.
Abstract 1679: Understanding the mechanism of Daple-PDGFRB gene fusion in leukemia
Authors: Ibarra Arnel Cardona , Ly Vivian , Ear Jason
Published: March 2024

Gene fusion between the scaffolding protein Daple and cell-surface receptor PDGFRB have been identified in several patients with leukemia. Patients with Daple-PDGFRB gene fusion are responsive to tyrosine kinase inhibitors, suggesting that the kinase domain on the receptor is active. How the Daple-PDGFRB gene fusion is activated and affects leukemia cells is poorly understood. It is hypothesized that the PDGFRB kinase domains found in gene fusion remain constitutively active, leading to hyperactivation of cell-signaling downstream of receptor tyrosine kinases (RTKs). To test this, identified Daple-PDGFRB mutants found in patients were ectopically expressed in cells and kinase activation were determined using a phospho-specific antibody. We demonstrated that indeed, the kinase domain on the gene fusion were active, and partake in rampant cell signaling. Because drug resistance is common in cancers such as leukemia, the implications from this work may uncover additional methods of therapeutic interventions for patients harboring these types of mutations.

Citation Format: Arnel Cardona Ibarra, Vivian Ly, Jason Ear. Understanding the mechanism of Daple-PDGFRB gene fusion in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1679.
Abstract 1729: SUV420H1 depletion reveals therapeutic potential in HPV-negative head and neck squamous cell carcinoma
Authors: Moshiri Arfa , Kim Sohyoung , Luff Marie , Saloura Vassiliki
Published: March 2024

Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) poses a significant clinical challenge with limited treatment options. SUV420H1, which encodes for a protein lysine methyltransferase (PMT) that methylates H4K20 (H4K20me3), is recurrently amplified in ~5% of HPV-negative HNSCC tumors and is the second most frequently amplified PMT in this disease, suggesting an oncogenic function in this subset of HPV-negative HNSCC patients. Gene set enrichment analysis using the TCGA database of HPV-negative HNSCC tumors revealed that SUV420H1 overexpressing tumors showed enrichment in the mitotic spindle related pathways, and repression of immune-related pathways. To assess whether SUV420H1 depletion affects the proliferation of HPV-negative HSNCC cells, CCK8 and colony formation assays were pursued after siRNA-mediated knockdown of SUV420H1 in five SUV420H1-overexpressing HPV-negative HNSCC cell lines and revealed a significant reduction in cell proliferation and colony forming capacity. SUV420H1 CRISPR knockout (KO) HPV-negative HNSCC human and mouse cell lines were generated. Proliferation and colony formation assays with the SUV420H1 KO cell lines are ongoing to validate the aforementioned phenotypes. To identify oncogenic mechanism of SUV420H1, genome-wide mapping of H4K20me3 after siRNA-mediated depletion or enzymatic inhibition of SUV420H1 (A-196) was pursued using CUT&RUN assays combined with RNA-seq in a SUV420H1 amplified cell line (SCC-151), and analysis is ongoing. Preliminary RNA-seq analysis revealed upregulation of a number of type I IFN response genes after siRNA-mediated knockdown of SUV420H1. In vivo mouse experiments using SUV420H1 KO mouse cell lines are planned in a syngeneic HPV-negative HNSCC flank mouse model (MOC1) with or without anti-PD-1 treatment with the goal to assess whether SUV420H1 depletion affects tumor growth and/or sensitizes cancer cells to anti-PD-1 therapy. These data provide preliminary support for the function of SUV420H1 as an oncogene in HPV-negative HNSCC.

Citation Format: Arfa Moshiri, Sohyoung Kim, Marie Luff, Vassiliki Saloura. SUV420H1 depletion reveals therapeutic potential in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1729.