bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2785: Beyond the role of transcobalamin 1 (TCN1) as a vitamin B12-binding protein in hepatocellular carcinoma
Authors: Muliawan Gregory Kenneth , Leung Carmen Oi Ning , Lee Terence Kin Wah
Published: March 2024

Hepatocellular carcinoma (HCC) is a common form of primary liver cancer and is associated with poor prognosis. Although sorafenib and lenvatinib have shown promising results in HCC treatment, their clinical benefits for patients have been limited owing to drug resistance and tumor relapse. This may be associated with the presence of tumor-initiating cells (T-ICs), which require further investigation to identify the key pathways or molecules involved in their regulation. Through in vitro enrichment of liver T-IC populations via a series of sphere passages under treatment with chemotherapeutic drugs, subsequent bulk RNA sequencing analysis identified a vitamin B-12 binding protein, transcobalamin 1 (TCN1), as the most upregulated molecule in such enriched T-IC populations. This result was consistent with the enrichment profiles of the stem cell signature in TCN1High HCC samples in the clinical dataset.

TCN1 is a secretory protein expressed at high levels in salivary glands and glandular cells; however, its function beyond its native role as a vitamin B12-binding protein in the regulation of T-ICs is unclear. Using lentiviral shRNA overexpression and knockdown approaches, we identified a critical role for TCN1 in regulating the properties of T-ICs, including self-renewal, invasiveness, tumorigenicity, and drug resistance, through in vitro and in vivo assays. Clinically, we also observed a substantial increase in TCN1 mRNA signatures in HCC samples, which was associated with poor prognosis of HCC patients. By analyzing the TCGA-LIHC dataset with confirmation by immunoprecipitation, we report, for the first time, that TCN1 directly interacts with integrin subunit alpha 3 (ITGA3), thereby regulating the Notch signaling pathway. Furthermore, ELISA results revealed the presence of the secretory form of TCN1 in HCC cells, which exerts a T-IC-enhancing effect on HCC cells via autocrine regulation.

In conclusion, our study provides novel insights into how TCN1 functions in HCC via a non-canonical mechanism, which goes beyond its conventional role as a vitamin B12-binding protein and uncovers a potential novel therapeutic target for HCC.

Citation Format: Gregory Kenneth Muliawan, Carmen Oi Ning Leung, Terence Kin Wah Lee. Beyond the role of transcobalamin 1 (TCN1) as a vitamin B12-binding protein in hepatocellular 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 2785.
Abstract 2786: Seizure related 6 homolog (SEZ6), a cancer stem cell-specific secreted protein, is a novel GBM therapeutic target
Authors: Mukherjee Arani , Sengupta Shreoshi , Chowdhury Abhishek , Srivastava Anand , Somasundaram Kumar
Published: March 2024

Cancer Stem Cells (CSCs) are known to initiate a tumor, resist chemotherapy and radiotherapy, and also cause tumor recurrence. A deeper understanding of CSC biology is needed for a more effective therapeutic response. An integrative study of regulated transcriptome of human patient-derived Glioma Stem-like Cells (GSCs) using Gene Ontology (GO) analysis and Gene Set Enrichment Analysis (GSEA) identified a potential role for GSC in tumor angiogenesis. We chose Seizure Related 6 Homolog (SEZ6), one of the Wnt target genes, for further study, as its downregulation inhibited the potential of GSCs to induce angiogenesis without impacting GSC growth. SEZ6, a transmembrane protein, is shed upon cleavage by Beta-site APP cleaving enzyme 1 (BACE 1) and is known to be involved in normal neuronal functions; however, its role in cancer has not been studied yet.

SEZ6 silencing inhibited GSC-initiated tumor growth in an intracranial orthotopic mouse model due to reduced tumor angiogenesis and increased the mouse survival. Pre-treatment with a pharmacological inhibitor of BACE1 (BACE1i) prevented SEZ6 shedding from GSCs and inhibited the ability of the GSC-secretome to induce angiogenesis. Further investigations are ongoing to establish the use of BACE1i or BACE1-silencing as a novel therapeutic strategy to inhibit SEZ6-mediated tumor angiogenesis.

We also carried out a protein array-based investigation, which revealed that SEZ6 induced IL8 in endothelial cells to induce angiogenesis. Additional investigation showed that SEZ6 activated the TGFβ pathway in endothelial cells to induce IL8. SEZ6 interacted with TGFβRII as seen by confocal microscopy and co-immunoprecipitation. Generation of various deletion mutants of SEZ6 revealed that three Sushi [also known as complement control protein (CCP) module or the short consensus repeat (SCR)] domains, 3, 4, and 5, interacted with TGFβRII. Molecular docking and dynamic simulation studies identified Sushi-3 domain as the mediator of the interaction between SEZ6 and TGFβRII extracellular domain. Based on the above results, experiments are underway to design specific inhibitors of this interaction. We are also developing an improved BACE1 inhibitor that would inhibit the shedding of SEZ6 into the extracellular milieu to inhibit SEZ6 functions.

Thus, this study establishes that GSC-secreted SEZ6 activates the TGFβ pathway in endothelial cells to induce tumor angiogenesis and proposes a novel therapeutic target.

Citation Format: Arani Mukherjee, Shreoshi Sengupta, Abhishek Chowdhury, Anand Srivastava, Kumar Somasundaram. Seizure related 6 homolog (SEZ6), a cancer stem cell-specific secreted protein, is a novel GBM therapeutic target [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 2786.
Abstract 2790: Hematopoietic stem cells tropism and conditioned media for targeting the triple negative breast cancer stem cells and associated mechanism: An in vitro study
Authors: Mallick Sumit , P Sudheer Shenoy , Bose Bipasha
Published: March 2024

Triple-negative breast cancers are the most challenging to treat with targeted therapy because of their non-expression of cell surface receptors, unlike other types of breast cancer. However, conventional therapies such as chemotherapy, radiotherapy, and surgery are standard treatment modalities. Moreover, drug encapsulation strategies to deliver drugs at the targeted site have been sought-after alternatives. Hematopoietic Stem Cells (HSCs) have unique features to migrate towards inflammatory sites and other body organs during any injuries and release various cytokines, which lead to interaction with molecular pathways and results in reduced inflammation and tissue injuries. From this characteristic, we have hypothesized that hematopoietic stem cells can show tumor tropism and modulate the metabolic alteration in cancer and cancer stem cells, mainly focusing on Triple Negative Breast Cancer Stem Cells (TNBC CSCs). In this study, we have sorted both cancer stem cells and hematopoietic stem cells from TNBC and hematological cell lines, respectively. Sorted HSCs were cultured in specific conditions to prepare conditioned media. Then, both cell’ types were used for co-cultured assay and conditioned media cultured with TNBC-CSCs were performed. Metabolomics studies were performed to decipher the molecular interactions and TNBC-CSCs metabolic alterations. The result indicates that the HSCs are showing positive tropism towards TNBC stem cells. Furthermore, HSCs-derived conditioned media (CM) interact with the cell cycle pathway by downregulating the CDK genes and causing DNA breakage in the TNBC-CSCs. Moreover, the Metabolomics studies further confirmed that HSCs CM-derived cytokines interact with various pathways, dysregulating the TNBC-CSCs proliferation and altering the metabolic activities.

Citation Format: Sumit Mallick, Sudheer Shenoy P, Bipasha Bose. Hematopoietic stem cells tropism and conditioned media for targeting the triple negative breast cancer stem cells and associated mechanism: An in vitro study [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 2790.
Abstract 2840: Development, characterization, and validation of a systemic human mantle cell lymphoma model utilizing luciferase expressing Jeko-1 tumor cells
Authors: Harris David , Wong Andrew , Rainbolt Elizabeth , Westrick Nicole , Avery Justin , Kolb Joseph , Vunnava Aswani , Ward Zachary , Hall Chassidy , John Bincy
Published: March 2024

Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphoma that is clinically and biologically heterogenous. MCL murine models are scarce and reliable in vivo models are necessary for pre-clinical testing and identification of improved novel therapeutics. To address this need, we established and characterized a human MCL in vivo model by systemic implantation of the luciferase-expressing Jeko-1 cell line (Jeko-1-luc) into immunodeficient NCG mice. Tumor growth kinetics was evaluated by testing multiple cell inoculums, and disease progression was monitored by in vivo serial bioimaging, three-dimensional optical imaging (3D), and ex-vivo endpoint analyses. Jeko-1-luc tumor growth profile revealed moderate latency with a median survival of 40 days post implant. Subsequently, we utilized 3D optical imaging and ex-vivo imaging that confirmed the localization of tumor growth predominantly in lungs, spleen, and skeletal regions. Additionally, we determined the surface expression of selected B-cell lineage markers including CD19, CD20, and CD22. Next, we evaluated the efficacy and effect on survival in response to various standard of care agents including Vincristine, Rituximab, Palbociclib, Temsirolimus, and Cyclophosphamide. The quantification of tumor burden by in vivo and ex-vivo bioluminescent imaging demonstrated that the highest therapeutic activity and overall survival was achieved by Palbociclib and Vincristine. Flow cytometric analyses further demonstrated that tumor cells obtained from blood and selected tissues including spleen and bone marrow showed reduced tumor burden in treatment groups relative to the control group. Taken together, these results illustrate the translational value of the Jeko-1-luc model and provides a platform for evaluating novel treatment modalities for mantle cell lymphoma.

Citation Format: David Harris, Andrew Wong, Elizabeth Rainbolt, Nicole Westrick, Justin Avery, Joseph Kolb, Aswani Vunnava, Zachary Ward, Chassidy Hall, Bincy John. Development, characterization, and validation of a systemic human mantle cell lymphoma model utilizing luciferase expressing Jeko-1 tumor 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 2840.
Abstract 2849: Palbociclib as a novel therapeutic for desmoplastic small round cell tumor
Authors: Magrath Justin W. , Sampath Shruthi Sanjitha , Flinchum Dane A. , Hartono Alifiani B. , Goldberg Ilon N. , Boehling Julia R. , Savkovic Suzana D. , Lee Sean B.
Published: March 2024

Desmoplastic small round cell tumor (DSRCT) is an aggressive, pediatric cancer caused by the EWSR1-WT1 fusion oncoprotein. Targeted therapies are lacking and the current standard of care, multimodal therapy, is insufficient, leading to a 5-year survival rate of less than 25%. While the EWSR1-WT1 oncoprotein is critical to growth in vitro, its importance has yet to be examined in vivo. Further, the set of EWSR1-WT1 downstream targets critical to oncogenesis and the mechanism by which the two EWSR1-WT1 isoforms regulate them remain underinvestigated. Here we generate a toolkit of DSRCT cell lines that deplete EWSR1-WT1 in a doxycycline (dox)-inducible manner. This toolkit enabled us to establish the essentiality of EWSR1-WT1 not only in vitro but also for the first time in vivo. Using this toolkit, we performed RNA sequencing on four DSRCT cell lines and established the most comprehensive analysis of EWSR1-WT1 downstream targets to date. We discovered novel mechanistic insights into EWSR1-WT1 functionality including the uniqueness of its transcriptional alterations as compared to native WT1, the direct role of EWSR1-WT1 binding in gene upregulation, and the dominant role of the E-KTS isoform in transcription. Given the rarity of DSRCT, we focused on downstream targets with existing inhibitors and perform a drug screen that identified the CDK4/6 inhibitor palbociclib as a novel DSRCT therapeutic. We found that the E-KTS isoform of EWSR1-WT1 binds to the CCND1 promoter, leads to CCND1 expression, and stimulates DSRCT growth through the CCND-CDK4/6-RB axis. Palbociclib treatment conversely reduced DSRCT growth by preventing retinoblastoma phosphorylation and blocking the transition from G1 to S phase. Palbociclib treatment was effective not only in vitro but also in vivo where it significantly reduced tumor growth in two xenograft models of DSRCT. Given these novel findings and palbociclib’s previous approval by the FDA for the treatment of breast cancer, we advance palbociclib as an exciting DSRCT therapy that warrants urgent clinical investigation.

Citation Format: Justin W. Magrath, Shruthi Sanjitha Sampath, Dane A. Flinchum, Alifiani B. Hartono, Ilon N. Goldberg, Julia R. Boehling, Suzana D. Savkovic, Sean B. Lee. Palbociclib as a novel therapeutic for desmoplastic small round cell tumor [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 2849.
Abstract 2852: Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells
Authors: Weichert-Leahey Nina , Berezovskaya Alla , Zimmerman Mark , Abraham Brian J. , Durbin Adam W. , Look A Thomas
Published: March 2024

Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system derived from migratory neural crest cells that have committed to become sympathetic neuroblasts, but their definitive differentiation is blocked. Neuroblastoma cell identity depends on the adrenergic core regulatory circuit (CRC) of transcription factors that collaborate with MYCN to drive cell proliferation and the oncogenic gene expression program. Retinoic acid is used as differentiation therapy for patients with high-risk neuroblastoma, with the goal to block proliferation and drive sympathetic neuronal differentiation. We recently showed that retinoic acid re-organizes the enhancer landscape of neuroblastoma and promotes a new retino-sympathetic cell state characterized by MYCN downregulation, proliferative arrest, and sympathetic neuronal differentiation. However, the effects of retinoic acid on epigenetic rewiring of the adrenergic CRC and suppression of MYCN expression are reversible in vitro and in vivo, leading to tumor re-growth when the retinoic acid is withdrawn. Therefore, we sought to determine if retinoid-induced differentiation can be forced to progress to irreversible neuronal maturation by the addition of inhibitors of epigenetic modifying enzymes. Here, we conducted a drug screen of epigenetic modifying drugs alone and in combination with retinoic acid to determine their effects on neuroblastoma cell growth and differentiation. In this screen, we found that an inhibitor of the histone H3K23 acetyltransferases KAT6A/B, PF-9363, synergistically inhibits neuroblastoma cell growth in combination with retinoic acid. Moreover, retinoid-resistant neuroblastoma cell lines can be sensitized to retinoic acid when combined with a KAT6 inhibitor. Importantly, this combination treatment renders the differentiated cell state irreversible, such that it is maintained after retinoic acid is withdrawn, with continued suppression of the adrenergic CRC and MYCN expression. In conclusion, the retino-sympathetic differentiated cell state induced by retinoic acid in neuroblastoma becomes irreversible when the cells are also treated with an inhibitor of the KAT6A/B histone H3K23 acetyltransferases, implicating the essential role of these enzymes in restoring the proliferative immature progenitor phenotype in adrenergic neuroblastoma cells.

Citation Format: Nina Weichert-Leahey, Alla Berezovskaya, Mark Zimmerman, Brian J. Abraham, Adam W. Durbin, A Thomas Look. Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma 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 2852.
Abstract 2876: Spatial immune cell atlas predicts the sensitivity to neoadjuvant PD-1 blockade treatment combined with radiotherapy in resectable esophageal squamous cell carcinoma
Authors: Yin Xiaoyang , Xu Kailun , Zheng Shu , Li Baosheng
Published: March 2024

Combination of radiotherapy and immunotherapy in neoadjuvant treatment seems a promising approach in resectable esophageal squamous cell squamous (ESCC). The clinical study of radiotherapy combined with PD-1 blockade therapy for neoadjuvant treatment of resectable ESCC shows good result. In order to investigate the underlying mechanism of the effectiveness of radiotherapy combined with immunotherapy in ESCC, we collected the pre and post neoadjuvant therapy samples from 5 responders and 5 non-responders ESCC patients and performed multiplexed tissue imaging using 28 markers. Using the detection measurements exported from QuPath, we performed the unsupervised clustering with Seurat and defined 20 cell types. Scimap was used to calculate the distance between different cell types. We found that CD4+ T, CD8+ T, cytotoxic CD8+ T cells and M1 macrophages showed an increase after treatment in both responders and non-responders. Tregs were decreased after neoadjuvant treatment in responder while there was no significant decrease in non-responder. And the distance between Tregs and cancer cells were increased in responders. Collectively, we provide an approach for investigating the underlying mechanism of the sensitivity to neoadjuvant PD-1 blockade treatment combined with radiotherapy in resectable ESCC using spatial multiplexed imaging.

Citation Format: Xiaoyang Yin, Kailun Xu, Shu Zheng, Baosheng Li. Spatial immune cell atlas predicts the sensitivity to neoadjuvant PD-1 blockade treatment combined with radiotherapy in resectable esophageal 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 2876.
Abstract 2878: Hyperthermia increases the efficacy of aminolevulinic acid-mediated photodynamic therapy in human osteosarcoma cells
Authors: Baugher Paige J.
Published: March 2024

Osteosarcoma is a malignant osteoid tumor that arises from mesenchymal cells exhibiting osteoblastic differentiation. Common treatments for this neoplasm include limb-salvage surgery and/or amputation of the affected limb, either of which can substantially decrease quality of life. Therefore, other, less invasive treatments should be explored. Photodynamic therapy (PDT) represents a more targeted, less invasive treatment possibility for osteosarcoma. Previous data from my laboratory demonstrated that aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) can be used to induce significant cell death in human osteosarcoma MG-63 cells in vitro. However, delivering high doses of drugs to deep osteosarcoma tumors could prove difficult, so we aim to explore the use of combination therapies to increase the efficacy of ALA-PDT in human osteosarcoma cells. Previous studies have shown that hyperthermia (increased temperature) can induce cell death in cancer cells. Furthermore, data suggest that combining PDT with hyperthermia can induce cell death in cancer cells synergistically by activating differential death-inducing pathways. However, hyperthermia in combination with ALA-PDT has not been explored in human osteosarcoma. Therefore, we aim to investigate the possibility that hyperthermia in combination with ALA-PDT can increase cell death in human osteosarcoma cells compared to ALA-PDT alone. Data from our lab show that compared to normal body temperature (37°C), cell death in MG-63 human osteosarcoma cells was not significantly increased by mild hyperthermia alone (40°C and 43°C respectively), while moderate hyperthermic temperatures alone (46°C and 49°C) did significantly increase cell death. We also found that at moderately hyperthermic temperatures, ALA-PDT does not increase cell death any further compared to moderate hyperthermia alone. Furthermore, we found that at the mildly hyperthermic temperature of 40°C, cell death was not significantly increased any further by ALA-PDT compared to ALA-PDT alone. However, our data do show that at a temperature of 43°C, hyperthermia plus ALA-PDT increased cell death compared to ALA-PDT alone. Therefore, our data suggest that inducing mild hyperthermia could increase the efficacy of ALA-PDT in human osteosarcoma cells.

Citation Format: Paige J. Baugher. Hyperthermia increases the efficacy of aminolevulinic acid-mediated photodynamic therapy in human osteosarcoma 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 2878.
Abstract 2919: Targeting regulatory T cells to boost immunity against melanoma
Authors: Miao Wanying , Jin Yingai J. , Jain Vaibhav , Gregory Simon , Zhang Jennifer Y.
Published: March 2024

Tumor-infiltrated regulatory T-cells (Treg) restrict the function of effector T-cells and thereby promote tumor growth. Disrupting the immunosuppression of Treg cells presents an attractive strategy for cancer immunotherapy; However, the risk of autoimmunity, resulting from systemic Treg depletion, limits its therapeutic effectiveness. Here, we show that genetic depletion of Ube2n in Treg cells inhibits tumor growth which is correlated with a markedly enhanced infiltration of cytotoxic immune cells. Notably, the deletion of Ube2n in a fraction of Treg cells was sufficient to produce a long-lasting anti-tumor effect, without apparent systemic autoimmune responses. The antitumor effect persists following adoptive T-cell transfer to an otherwise immunodeficient mouse tumor model. Through flow cytometry, immunostaining and T-cell targeted single-cell sequencing analyses, we demonstrate that the loss of Ube2n dampens Treg suppressive function, as indicated by markedly increased numbers of cytotoxic CD8 T-cells in the tumor microenvironment (TME). Further, differentially expressed genes analyses show a downregulation of Mki67 and an upregulation of Ifng in Ube2n deficient Tregs, concomitant with amplification and activation of effector T-cells. Our findings uncover a hitherto unexplored role of Ube2n in orchestrating the differentiation and functions of Treg in TME, highlighting the therapeutic potential of targeting this pathway to enhance and reprogram anti-tumor immunity.

Citation Format: Wanying Miao, Yingai J. Jin, Vaibhav Jain, Simon Gregory, Jennifer Y. Zhang. Targeting regulatory T cells to boost immunity against melanoma [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 2919.
Abstract 2946: Characterizing the genomic landscapes of breast and lung tumors using cost-effective whole genome sequencing
Authors: Benjamin Hila , Soifer Ilya , Shem-Tov Doron , Levy Maya , Tuncay Islam Oguz , Oh Baek-Lok , Shin Jong-Yeon , Ju Young Seok , Lee Sangmoon , Barad Omer , Lipson Doron
Published: March 2024

Whole Genome Sequencing (WGS) has emerged as a pivotal tool for unraveling the intricate genomic alterations underlying cancer, enabling the detection of a broad spectrum of somatic changes in cancer genomes, including single nucleotide variants, insertions, deletions, copy number variations, and structural rearrangements. Characterizing somatic variants across the entire genome significantly benefits from novel cost-efficient sequencing platforms, such as UG100. In the current study, we aim to demonstrate the utility of UG100 WGS in characterizing the landscape of somatic events in breast and lung cancer.

Over 35 tumor-normal cancer sample pairs were profiled using UG100 WGS (mean tumor coverage: >100x; normal: >50x). Variant calling was done using a somatic deep-variant algorithm, optimized to UG100 data. Results were compared to Illumina sequencing and high concordance was observed, with over 97% of SNVs and over 93% of indels detected by Illumina identified also by UG100. UG100 sequencing was deeper and allowed for identification of a larger number of genomic variations, including known driver events in cancer-related genes. The somatic mutational signatures by UG100 were concordant with the Illumina platform, with a cosine similarity higher than 99%. Copy number variation and structural variation analysis was performed on all sample pairs, revealing a wide range of genomic aberrations.

The integration of genetic sequencing into routine clinical oncology practices has proven instrumental in identifying actionable mutations, predicting treatment responses, and monitoring minimal residual disease. Moving from targeted sequencing to WGS provides a more comprehensive and unbiased assessment of the entire genome, uncovering rare or unexpected mutations that might be missed by targeted approaches. WGS allows for the simultaneous evaluation of both coding and non-coding regions of the genome, providing a deeper understanding of the regulatory elements influencing gene expression. This broader scope enhances our understanding of cancer biology and contributes to a more accurate assessment of the tumor's functional landscape.

Citation Format: Hila Benjamin, Ilya Soifer, Doron Shem-Tov, Maya Levy, Islam Oguz Tuncay, Baek-Lok Oh, Jong-Yeon Shin, Young Seok Ju, Sangmoon Lee, Omer Barad, Doron Lipson. Characterizing the genomic landscapes of breast and lung tumors using cost-effective whole genome sequencing [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 2946.
Abstract 2972: Senescent macrophages drive tumorigenesis by limiting antitumor immunity
Authors: Prieto Luis I.
Published: March 2024

A big challenge in oncology is tumor heterogeneity, as the same type of tumor can manifest differently across patients. Our current cancer therapies focus on targeting malignant cells without considering how the tumor microenvironment influence tumorigenesis or changes in response to treatment. Therefore, identifying effective therapies that restrict tumor growth and progression while restoring optimal tissue and organ function is imperative. Here, we propose a potential strategy to delay the frequency of lung tumorigenesis and reduce progression by selectively targeting tumor-promoting senescent cells that arise with advancing age, prolonging antitumor immunity.

Using lung tumor-prone mice, we found that senescent cells, specifically senescent alveolar macrophages, accumulate early in hyperplastic lesions. Mechanistically, these macrophages upregulate the cell cycle regulator p16Ink4a; express the cell surface protein CXCR1; they are distinct from other established subsets; are sensitive to senolytic interventions; have a tumor-promoting secretome, including SPP1; and express immunosuppressive factors that modulate the antitumor immune response. Selective clearance of senescent macrophages attenuates adenoma development, indicating they, at least in part, promote tumorigenesis. Importantly, we observed alveolar macrophages with these properties accumulating in treatment-naive human early-stage lung adenocarcinoma in situ, indicating that the cellular senescence program in macrophages is a conserved etiology. We also found that senescent alveolar macrophages accumulate in otherwise naturally aged normal mouse lungs and their persistence correlates with increased inflammation. Collectively, our findings suggest that targeting senescent macrophages may constitute a potential therapeutic intervention to postpone or attenuate lung cancer during the early stages of the disease.

Citation Format: Luis I. Prieto. Senescent macrophages drive tumorigenesis by limiting antitumor immunity [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 2972.
Abstract 2994: MIR99AHG promotes ERα expression and ER-positive breast cancer cells proliferation via activating Hippo signaling
Authors: Wang Wende , Lin Danping , Lin Ziyang , Yan Yangyang , Qin Yusen , Liang Yuanke , Lin Haoyu , Zeng De
Published: March 2024

Background: Estrogen receptor (ER) positive breast cancer is the most common subtype of breast cancer. Elucidation of the regulatory network in estrogen receptor pathway is critical to develop and deliver potent targeted therapy for ER-positive breast cancer. The Mir-99a-let-7c cluster host gene MIR99AHG is a long non-coding RNA involving in a variety of tumor formation and disease progression processes. However, the biological functions of MIR99AHG in ER-positive breast cancer have not been reported.

Methods: The Western-blot, qRT-PCR, Immunofluorescence and fluorescence in-situ hybridization were used to identify potential target genes and regulatory pathway of estrogen receptor α (ERa), which is a key proliferative driver of ER-positive breast cancer cells. The cell proliferation assay, clone formation assay, xenograft tumor model and rescue assay were used to investigate the effect of differential expression of MIR99AHG on the proliferative capacity of ER-positive breast cancer cells MCF7 and T47D. In addition, correlation analysis was performed to verify the relation of the expression of MIR99AHG, LATS1, LATS2, CYR61, CTGF and ESR1 from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cohorts. And survival analysis was used to predict the prognostic impact of MIR99AHG in breast cancer patients.

Results: MIR99AHG expression was significantly higher in ER-positive breast cancer cells MCF7 and T47D than in ER-negative breast cancer cells SKBR3, MDA-MB-231 and BT549. The high expression of MIR99AHG predicted poor prognosis in ER-positive breast cancer patients. In vitro and in vivo assays demonstrated that MIR99AHG positively regulated the proliferation of MCF7 and T47D cells. Importantly, the Hippo signaling was identified to be down-stream mediators of MIR99AHG in modulating ERα transcription. Furthermore, XMU-MP-1, which targets the Hippo signaling upstream kinase MST1/2, was able to reverse the effect of MIR99AHG on MCF7 cell proliferation.

Conclusions: MIR99AHG up-regulates ERα expression and promotes proliferation in ER-positive breast cancer cells through activating Hippo signaling pathway. Targeting MIR99AHG/Hippo pathway is a potential strategy for the treatment of ER-positive breast cancer.

Citation Format: Wende Wang, Danping Lin, Ziyang Lin, Yangyang Yan, Yusen Qin, Yuanke Liang, Haoyu Lin, De Zeng. MIR99AHG promotes ERα expression and ER-positive breast cancer cells proliferation via activating Hippo signaling [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 2994.
Abstract 3025: Germline p53 R181 variants and DNA binding cooperativity in tumorigenesis
Authors: Moses Renyta , Hausler Ryan , Kelly Gregory , Indeglia Alexandra , Miller Sven , Karanicolas John , Murphy Maureen , Maxwell Kara
Published: March 2024

TP53 is the most frequently mutated gene in cancer, and its encoded protein p53 has many tumor-suppressive functions. There are several different classes of mutant p53 acquired in human cancer and inherited in cancer-prone families with Li-Fraumeni Syndrome, where individuals have an 80-90% increased risk of cancer. These include structural mutations that are generally (e.g R175H) or locally (e.g Y220C) misfolded, DNA contact mutations (e.g R273H); and oligomerization mutations (A347D). Cooperativity mutations affect binding of the p53 tetramer to DNA via disruption of a salt bridge formed by the negatively charged glutamic acid (E) 180 residue of one p53 monomer and the positively charged arginine (R) 181 residue of another. p53 “cooperativity” mutations at the R181 residue (R181H and R181C) have been increasingly identified in cancer-prone families undergoing genetic testing; however, the mechanism by which these variants disrupt p53 tumor suppression in humans is not understood. We show that the purified DNA binding domains of p53 variants R181H and R181C bind less cooperatively to the p53 binding site in the CDKN1A (p21) promoter, despite retaining wild-type levels of structural stability. RNA-sequencing of CRISPR knock-in colorectal and breast cancer cell lines shows reduced ability of R181H and R181C to transactivate a curated set of ~300 known p53 target genes. Upon treatment with p53 activating molecule Nutlin-3a, the R181-mutant cells fail to cell cycle arrest in the G1 phase and maintain high proliferative rates. Interestingly, we observe some residual apoptotic activity in R181H and R181C mutant cells treated with DNA-damaging agent 5-fluorouracil, despite losing the transactivation of proapoptotic p53 targets; this suggests that these mutants retain the p53 transcription-independent mechanism of apoptosis which is observed in other p53 variants such as P47S and A347D. These studies will define the pathogenicity of R181 variants and guide therapeutic intervention for patients that harbor these p53 mutations.

Citation Format: Renyta Moses, Ryan Hausler, Gregory Kelly, Alexandra Indeglia, Sven Miller, John Karanicolas, Maureen Murphy, Kara Maxwell. Germline p53 R181 variants and DNA binding cooperativity in 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 3025.
Abstract 3049: Similarities between ovarian cancer and endometriosis: The role of STAT3
Authors: Kloeckner Allison A. , Walker Sarah R.
Published: March 2024

Endometriosis is a painful gynecological disease that affects over 190 million women worldwide. This disease is classified by the presence of endometrial-like cells growing in and around the female reproductive system. Instead of lining the uterus, these ectopic cells shed off and cluster together forming lesions that attach to different surfaces of the female reproductive system. In particular, endometriosis cells interact with and form lesions within the mesothelial cells that line the peritoneum. There is currently no cure, and many of the therapies aiming to help with pain management come with unfavorable tradeoffs. Our research on ovarian cancer led us to investigate potential overlapping characteristics with endometriosis. Growing ovarian cancer cells in 3D causes spheroids to form which mimic the physical features of tumors and are also able to better represent cell-cell and cell-environment interactions. Analysis of the endometriosis cell lines 12Z and hEM3 demonstrated that they could also form spheroids. As both ovarian cancer cells and endometriosis cells interact with mesothelial cells, we wanted to determine if endometriosis spheroids were able to clear the mesothelial layer comparable to ovarian cancer spheroids. Evaluation of 12Z and hEM3 cell lines demonstrated that endometriosis spheroids were able to undergo mesothelial clearance. While the exact cause of endometriosis is not known, different proteins have been identified as important factors in the progression of endometriosis. The Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor involved in cell differentiation, proliferation, apoptosis resistance, angiogenesis, and metastasis. In endometriosis cell lines, STAT3 is constitutively active, which makes it a promising potential therapeutic target. To verify that STAT3 is important in endometriosis, siRNA was used to reduce the expression of STAT3 in two endometriosis cell lines, 12Z and hEM3. Both cell lines showed a decrease in cell viability. In addition, reducing the expression of STAT3 in the endometriosis cells appeared to decrease mesothelial clearance ability. Our lab previously identified statins as potential STAT3 inhibitors by using the Broad Institute’s CLUE tool. After statin treatment, 12Z and hEM3 spheroids showed a decrease in cell viability and clearance ability. Taken together, these data suggest that STAT3 plays a key role in endometriosis and that the repurposing of statins may provide a benefit to patients with endometriosis.

Citation Format: Allison A. Kloeckner, Sarah R. Walker. Similarities between ovarian cancer and endometriosis: The role of STAT3 [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 3049.
Abstract 3054: Investigating novel players involved in AR cistrome reprogramming
Authors: Gardner Ephraim Jeremiah
Published: March 2024

Advancements in the treatment of aggressive prostate cancer (PCa) have led to the development of second-generation AR-targeted therapeutics for advanced PCa. Unfortunately, many patients who initially respond to these treatments develop resistance and progress. Several mechanisms of resistance are being studied. We have focused on the roles of a reprogrammed AR cistrome from a canonical to a non-canonical cistrome in resistant disease. The premise of this study is based on previous studies by others and studies in our lab that suggest enrichment in non-canonical AR activity and adaptation to enzalutamide in advanced PCa cell lines and patient-derived xenografts. We aim to develop an ecosystem map of canonical and non-canonical AR activity to understand the drivers of therapeutic resistance and their vulnerabilities. We hypothesize that through combined transcriptomic/chromatin accessibility profiling at single-cell resolution and proteomics studies, we can holistically capture non-canonical AR activity. To provide invaluable information, as PCa is highly heterogeneous and not all cancer cells may be contributing to the resistance pathway that is of interest, we use castrate-resistant PCa (CRPC) and enzalutamide-resistant CRPC patient-derived xenograft (PDX) models and patient tissue samples as our disease models on our multi-omic assay platform to capture and study dynamic interplays and novel players driving a non-canonical AR cistrome and treatment resistance. Our preliminary data revealed potential transcriptional factor (TF) candidates that may modulate non-canonical AR cistrome. We verified the enrichment of these TF in PDX models of treatment-resistant PCa at the gene and protein level. Integration of our ongoing global proteomic studies will provide a holistic view of the contributions of the TF to the AR non-canonical switch.

Citation Format: Ephraim Jeremiah Gardner. Investigating novel players involved in AR cistrome reprogramming [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 3054.
Abstract 3062: Unraveling the role of RCC2 in glioblastoma by mutational analysis and a murine model of glioma
Authors: Ho Wayne L.H. , Or Penelope M.Y. , Chan Andrew M.
Published: March 2024

Glioblastoma (GBM) is a brain malignancy characterized by being highly aggressive, lethal, yet treatment modalities remain limited. Recent studies have implicated Regulator of Chromosome Condensation 2 (RCC2), a guanine nucleotide exchange factor (GEF) for RalA, as a potential regulator in glioblastoma pathogenesis. RCC2 has been shown to play a role in promoting glucose metabolism through BACH1-dependent upregulation of hexokinase II (HK2)(1). In this study, we investigated the functional mechanism of RCC2 in gliomagenesis using two complementary approaches. First, the relevance of RCC2 structural regions on its downstream functions were examined. By superimposing the three-dimensional crystal structure of RCC2 onto RCC1 (Regulator of Chromosome Condensation 1), a related GEF, we identified several unique regions within the RCC1-like domain of RCC2. A panel of point, substitution, and deletion mutants were generated, and pulldown experiments were performed to assess their relative binding affinity with BACH1. Additional assays to assess their relative ability to modulate the expression of HK2 and other cellular parameters will be discussed. The structure-functional characterization of distinct regions within RCC2 may identify unique sequences within RCC2 that could have relevance to glucose metabolism, cell proliferation and cell motility. Additionally, we have established a murine glioma model utilizing a Pten heterozygous mice expressing a GFAP-Cre-recombinase transgene (Gfap-cre;Ptenfl/+). Glioma were induced by stereotaxically injected lentiviral constructs expressing either with HRasV12 oncogene alone or together with RCC2. Neurological symptoms were monitored over a period of 7 months, and histopathological analysis was performed to evaluate the morphological and molecular features of the tumors. We showed that co-expression of HRasV12 and RCC2 exhibited earlier emergence of neurological manifests in both male and female groups (Log-rank test, p<0.05) when compared to HRasV12 alone. Overall, our study provides insights into both the structural mechanism of RCC2 in gliomagenesis through mutational analysis of RCC2 and the functional relevance through a murine model of glioma.

(1)Liu, T. et. al. Cancer Lett (2022) 549:215914.

Funding: This work is supported by a General Research Fund grant (#14113519) from the University Grants Committee of Hong Kong.

Citation Format: Wayne L.H. Ho, Penelope M.Y. Or, Andrew M. Chan. Unraveling the role of RCC2 in glioblastoma by mutational analysis and a murine model of glioma [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 3062.
Abstract 3099: A new HTS ELISA for monitoring NRAS activity to expedite drug discovery
Authors: Park Hyun Min , Weber Julie , Carlson Michelle , Jelinek Mary Anne
Published: March 2024

Oncogenic mutations in the KRAS, HRAS, and NRAS isoforms of the RAS gene family alter GTP binding and hydrolysis leading to errant over activation of the MAPK/ERK pathway. While the total of all such RAS mutations drives some 20-25% of all human cancers, each isoform tends to favor particular mutations that are cancer specific. Many evolving treatment strategies for these cancers target these mutant isoforms to minimize off target effects. NRAS mutations are seen in about one-fifth of cutaneous melanomas and patients with NRAS mutation melanomas have a poorer prognosis due to the high aggressiveness of RAS mutant tumors, a lack of efficient targeted therapies, or rapidly emerging resistance to existing treatments. Furthermore, there is no currently authorized targeted treatment for NRAS-mutated melanoma. As a result, there is a growing need for assays which can quantify activated NRAS. Here we describe the development of a highly specific and sensitive ELISA for determining the active NRAS protein in high throughput compatible (HTS) format.

A NRAS specific recombinant antibody was developed via cloning of immunoglobulin variable regions from antigen positive B cells isolated by FACS from rabbits immunized with a NRAS peptide corresponding to a region of low homology between the RAS isoforms. Immunoblotting and a bead-based multiplex ELISA with all three RAS isoforms were used to confirm antibody specificity with recombinant HRAS, KRAS, and NRAS proteins over a wide concentration range.

The new immunoassay enables in-well lysis of cells cultivated in 96-well plate. Activated NRAS in the lysate is collected on the test plate and detected using the NRAS specific antibody via chemiluminescence. We anticipate that this assay will further the discovery of novel NRAS specific therapeutics. EGF treatment of MCF7 cells demonstrated activation in cells expressing wildtype NRAS in preliminary assay validation. Evaluation of assay performance in mutant NRAS melanoma cell lines will be presented.

Citation Format: Hyun Min Park, Julie Weber, Michelle Carlson, Mary Anne Jelinek. A new HTS ELISA for monitoring NRAS activity to expedite drug discovery [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 3099.
Abstract 3115: Systematic analysis of gene mutations and therapeutic efficacy via ex vivo hematological vitroscreen platform
Authors: Chou Fu-Ju , Cairo Stefano , Gilardi Mara , Andar Abhay , Abarca-Heidemann Karin , Mancini Maria
Published: March 2024

Acute myeloid leukemia (AML) is a common hematological malignancy found in adult patients, and FMS-like tyrosine kinase 3 gene (FLT3) mutation occurs in about 30% of patients. In 2019, a phase 3 clinical trial suggested that gilteritinib treatment resulted in significantly longer survival than salvage chemotherapy in relapsed or refractory FLT3-mutated AML patients. Targeting FLT3-mutants in AML has become one of the important therapeutic strategies in this decade. The ex vivo hematological VitroScreen platform provides a powerful tool to evaluate the performance of FLT3 inhibitors and other test articles. With Champions Oncology’s primary hematological repository (https://www.championsoncology.com/hematological-vitroscreen) and proprietary LUMIN Bioinformatics platform, we classified models based on patient responses, clinical annotation, NGS data, and cell surface markers. In this Ex vivo study, we tested gilteritinib in 30 AML patients, and 8 of models were identified as FLT-3 mutants’ carrier. Fold changes of cell viability after gilteritinib treatment were assessed based on quantitation of the ATP present. FLT3-mutants models showed significantly higher sensitivity to gilteritinib than FLT3-WT models (IC50, 132.56 nM vs. 234.16nM) in a 6-day assay. Only 25% of models in the FLT3-mutants group had higher IC50 than the average of all AML models (n=28, IC50 = 204.05 nM), compared to 53% in the FLT3-WT group. Our preclinical data highly match the observations in previous clinical trials, which suggest that VitroScreen is a reliable high throughput tool for drug development of drug resistant cancers.

Citation Format: Fu-Ju Chou, Stefano Cairo, Mara Gilardi, Abhay Andar, Karin Abarca-Heidemann, Maria Mancini. Systematic analysis of gene mutations and therapeutic efficacy via ex vivo hematological vitroscreen platform [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 3115.