bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1340: Modulation of the natural killer cell immune response to tumor with a synthetic tumor -immune cell agonist, NK-TICA™
Authors: Dufort Fay , Ezell Tucker , Rezvaya Alexandra , Ho Kathleen , Leitheiser Christopher , Brandish Philip , McDonnell Kevin , Keen Nicholas
Published: March 2024

Natural Killer (NK) cells are highly responsive cytotoxic immune cells of the innate immune system with well characterized anti-tumor properties. Their ability to directly kill malignant cells and elicit an adaptive immune response makes them a promising candidate for a precision guided immunotherapy in oncology. Bicycle® peptides are small (ca.1.5kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have applied the Bicycle® platform technology to discover and evaluate a new class of fully synthetic molecules termed NK tumor immune cell agonists (NK-TICA™). The NK-TICA™ consists of chemically coupled Bicycle® peptides that bind specifically to the key activating receptor, NKp46, and to tumor antigens, that results in highly potent, antigen-dependent receptor activation and NK cell function. We demonstrate potent, selective binding of our Bicycle® peptides to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function in vitro. With Bicycle’s novel NK-TICA™ compound, we demonstrate the engagement of NK cells, the specific activation and function of NK cells, and enhanced tumor cytotoxicity in a tumor target- and dose-dependent manner. In conclusion, NK-TICAs drive NK cell-mediated tumor cell killing and cytokine production in vitro and as such have the potential to catalyze the development of durable anti-tumor immunity in tumor types not well served by current therapies. We hypothesize that utilization of Bicycle® NK-TICA™ as a multifunctional immune cell engager will promote the modulation and anti-tumor activity of peripheral and intra-tumoral NK cells to solid tumors.

Citation Format: Fay Dufort, Tucker Ezell, Alexandra Rezvaya, Kathleen Ho, Christopher Leitheiser, Philip Brandish, Kevin McDonnell, Nicholas Keen. Modulation of the natural killer cell immune response to tumor with a synthetic tumor -immune cell agonist, NK-TICA™ [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 1340.
Abstract 1343: BSI-111, a highly selective anti-CD16a monoclonal antibody with potent agonist activity for building a NK cell engager platform
Authors: Liu Jinyu , Dai Wenwen , Li Hongyan , Liu Xiaodong F. , Li Jun , Xia Shukai , Lyu Qun , Davis Hugh M. , Chen Mingjiu , Peng Zeyu
Published: March 2024

Background: Natural killer cells (NK) are innate lymphocytes endowed with the ability to recognize and kill cancer cells. CD16a, a well-characterized activating receptor predominantly expressed on NK cells, is a low-affinity receptor for the IgG Fc domain and is crucial for stimulating NK cell-mediated tumor cell killing. Unlike CD16a, CD16b is mainly expressed on granulocytes as a GPI-anchored receptor and is not involved in the lysis of tumor cells although CD16a and CD16b share 97% sequence identity in their extracellular domains. Due to the high homology of extracellular domains of CD16a and CD16b, it is extremely challenging to develop CD16a-specific agonistic antibodies without binding CD16b+ granulocytes, the most abundant subset in leukocytes. Here we report the development of an anti-CD16a-specific monoclonal antibody with potent NK cell activation.

Experimental procedures: Humanized mice were immunized with recombinant CD16a-ECD-Fc. The Biosion proprietary H3 (High-throughput, High-content and High-efficiency) antibody screening platform was used to identify an anti-CD16a monoclonal antibody lead candidate - BSI-111. The target binding specificity, binding activity, and affinity of BSI-111 were evaluated by protein-based ELISA, cell-based FACS and Biacore-based SPR. A cell-based agonist reporter assay and a primary NK cell activation assay were used to evaluate the bioactivity of BSI-111.

Summary: BSI-111 is a fully human monoclonal antibody with the following critical properties: (1) specifically binds to CD16a without recognizing CD16b; (2) binds the two allelic variants of CD16A - 158F and 158V with similar high affinities; (3) shows cross-reactivity to cynomolgus CD16a; (4) can significantly activate NFAT signaling in a cell-based reporter assay; (5) exhibits strong activity on activating primary NK cells; and (6) has potentially longer half-life and silenced Fc-effector function through Fc-engineering.

Conclusion: BSI-111 demonstrates great biophysical properties and functional characteristics, supporting the development of anti-CD16a-based NK cell engagers for potential benefit of cancer patients.

Citation Format: Jinyu Liu, Wenwen Dai, Hongyan Li, Xiaodong F. Liu, Jun Li, Shukai Xia, Qun Lyu, Hugh M. Davis, Mingjiu Chen, Zeyu Peng. BSI-111, a highly selective anti-CD16a monoclonal antibody with potent agonist activity for building a NK cell engager 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 1343.
Abstract 1345: NAV-201, an orally bioavailable small molecule NK cell activator enhancing antibody-mediated cancer therapies
Authors: Nicolaides Nicholas C. , Grasso Luigi , Kline J. Brad
Published: March 2024

Tumors employ a number of mechanisms to suppress a patient’s immune anti-tumor responses. Recently, we uncovered an immunosuppression mechanism that tumors employ to avoid humoral immune responses that involves tumor-produced proteins, called Humoral Immuno-Oncology (HIO) factors. These HIO factors are able to suppress antibody-mediated tumor cell killing via direct binding to antibodies that alter their Fc domain interactions with the C1q protein to suppress complement dependent cytotoxicity (CDC) and reduce antibody engagement with CD16a Fc receptors on immune-effector cells, including NK cells, to suppress antibody dependent cellular cytotoxicity (ADCC). HIO factor immunosuppression of ADCC appears to involve the reduced binding affinity of antibody to CD16a Fc receptors. In an attempt to identify agents that may enhance antibody-CD16a binding affinity to overcome HIO factor immunosuppression, we sought to identify agents that can restore this interaction by screening natural small compound libraries via an IgG1-CD16a based ELISA assay. One lead compound was identified that was subsequently optimized by structural activity relationship analysis. This compound, named NAV-201, specifically binds CD16a and enhances the affinity of antibody binding to both the low affinity CD16a-158F and high affinity CD16a-158V allotypes. Moreover, we found that NAV-201 can also bind to the mouse CD16a ortholog and enhance its antibody binding affinity, making NAV-201 suitable for efficacy testing in mouse models. In vivo, NAV-201 was found to be well tolerated up to at least 120 mg/kg when administered orally and daily to mice, have a serum half-life of 18 hours, enhance systemic levels of the NK cell produced INF-γ and TNF-α cytokines, and enhance the therapeutic efficacy of antibodies in mouse xenografts. Together, these data support the further development of NAV-201 for human testing to improve and/or enhance the efficacy of antibody-based therapies in HIO factor positive cancers.

Citation Format: Nicholas C. Nicolaides, Luigi Grasso, J. Brad Kline. NAV-201, an orally bioavailable small molecule NK cell activator enhancing antibody-mediated cancer therapies [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 1345.
Abstract 1374: Targeting CD47/SIRPɑ axis in urologic cancer triggers enhanced phagocytosis of tumor cell and induces tumor growth inhibition
Authors: Shen Fan , Praharaj Monali , Lee Alex J. , Nirschl Thomas R. , Wang Xiaoxu
Published: March 2024

Signal regulatory protein-α (SIRPα; CD172a or SHPS-1) is a transmembrane molecule that highly expressed on myeloid population. Binding of CD47 (integrin-associated protein, IAP) to SIRPα inhibits phagocytosis in macrophages, which acts as a mechanism of maintaining homeostasis. Tumor cells however, often time upregulate CD47 to evade immune response. Using publicly available dataset, we found that human prostate cancer cells upregulate expression of both CD47 and calreticulin, a “eat me” signal when engaged with Low density lipoprotein receptor-related protein 1 (LRP1). In addition, macrophages, monocytes and dendritic cells in human prostate cancer do have relatively high level of SIRPɑ and LRP1 expression. This finding provides the rationale of targeting CD47/SIRPɑ axis in prostate cancer to shift the balance between pro-phagocytosis and anti-phagocytosis signaling. We then moved to RM1 and B6Cap, two syngeneic prostate cancer models in immunocompetent mice. Significant tumor growth inhibition was seen in both models with anti SIRPɑ blocking antibody. Furthermore, we observed in vitro that CD47 or SIRPɑ blockage led to enhanced PC3 cell line phagocytosis by human monocyte derived macrophages (HMDMs). Same finding was found using mouse bone marrow derived macrophages (BMDMs). We then utilized ovalbumin expressing MB49 bladder cancer cell line and cocultured those with BMDMs. CD8+ T cell from OT1 mice exhibited higher expression of activation makers when cocultured BMDMs treated by either anti CD47 or SIRPɑ antibody, indicating that enhanced phagocytosis subsequently increases antigen cross presentation in macrophages. In summary, we identified that CD47 is overexpressed in human prostate cancer; blocking CD47/SIRPɑ did inhibit tumor growth through enhanced phagocytosis and antigen presentation.

Citation Format: Fan Shen, Monali Praharaj, Alex J. Lee, Thomas R. Nirschl, Xiaoxu Wang. Targeting CD47/SIRPɑ axis in urologic cancer triggers enhanced phagocytosis of tumor cell and induces tumor growth inhibition [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 1374.
Abstract 1385: Crosstalk between hepatocyte and myeloid-specific IL17 signaling in colorectal cancer metastasis
Authors: Chojnacka Katarzyna , Kamen Dan , Reyes Katrina M. , Tomizawa Hana , Grivennikov Sergei I.
Published: March 2024

Advanced colorectal cancer (CRC) is resistant to current immunotherapies and prone to metastasis. Chronic inflammation mediated by cytokines such as interleukin (IL)17 activates the oncogenic pathways in cancer cells, promoting sporadic and inflammation-associated CRC. Increased levels of IL17A in early stages of CRC portends poor prognosis indicating its potential involvement into metastasis. We and others previously showed that IL17A signaling is required for early CRC tumorigenicity in a variety of models and that in early CRC, its ability to signal to epithelial cells is essential. What remains unknown is whether IL17 signaling to other cell types within the tumor microenvironment (TME) is essential to control tumor progression and metastasis. Using a model of colitis-associated cancer (CAC) we found that “late” IL17A neutralization decreases tumor burden, possibly acting through regulation of myeloid cells and chemokine network essential for the control of immunosuppressive TME. Next, using models of colorectal cancer liver metastasis (CRLM) including intraportal cell injection and cecum transplantation of organoids with distinct set of oncogenic mutation we found that IL17 neutralization reduces metastasis. FACS analysis revealed that IL17A promotes the recruitment of tumor-associated macrophages and other key myeloid subsets into the metastatic liver environment. Interestingly, ablation of IL17RC (receptor) in myeloid cells only (using Il17rcf/f -LysMCre mice) led to reduction in metastasis and reshaping of metastatic myeloid cell compartments. Meanwhile, IL17 signaling in hepatocytes was required for early metastasis and pre-metastatic niche formation, as the ablation of IL17RC in hepatocytes (using Il17rcf/f -AlbCre mice) led to a reduction of metastatic numbers but not growth. This coincided with reduced neutrophil recruitment and enhanced activation of cytotoxic innate lymphoid cells and CD8 T lymphocytes, indicating the role of hepatocyte specific IL17 signaling in blunting anti-tumor immunity. Our findings illuminate the role of inflammatory cytokine IL17 into CRC progression and metastasis and imply its distinct cell type specific role within the metastatic liver TME.

Citation Format: Katarzyna Chojnacka, Dan Kamen, Katrina M. Reyes, Hana Tomizawa, Sergei I. Grivennikov. Crosstalk between hepatocyte and myeloid-specific IL17 signaling in colorectal cancer 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 1385.
Abstract 1386: CD1d-restricted iNKT cells promote liver metastasis from colon cancer
Authors: Nater Marc , Lucchiari Giulia , Brügger Michael , Valenta Tomas , Cecconi Virginia , Forni Geo , Benke Stephan , Broek Maries van den
Published: March 2024

The liver is a prominent site for metastasis, yet little is known about the role of liver immunity in antimetastatic defense. Compared to other organs, the liver harbors a unique immune environment characterized by the abundance of innate cell lineages, including CD1d-restricted invariant natural killer T (iNKT) cells. To investigate the influence of iNKT cells on metastasis, we established an orthotopic model of colorectal cancer (CRC) with spontaneous dissemination to the liver. We induce the primary tumor by implanting CRC organoids into the colon mucosa of syngeneic C57BL/6 mice, resulting in hepatic macrometastases within 5 weeks. We observed that liver metastasis hardly occurred in mouse strains lacking iNKT cells (Cd1d−/− and Traj18−/− mice), while the growth of the primary tumor was comparable to that in wild-type mice. These results indicate a tumor-promoting function for hepatic iNKT cells. We uncovered that hepatic iNKT cells respond to disseminating cancer by producing IL-4 and IL-13, two cytokines known to activate hepatic stellate cells (HSCs) into fibrogenic myofibroblasts. Indeed, we saw rapid activation of HSCs and deposition of extracellular matrix upon arrival of cancer cells in the liver of wild-type but not iNKT cell-deficient mice. We propose that hepatic iNKT cells contribute to the formation of a supportive niche for disseminated tumor cells by activating HSCs early in the development of hepatic metastasis. Taken together, our findings highlight an essential tumor-promoting role for iNKT cells during the development of liver metastasis and contribute to our understanding of immune regulation of metastasis.

Citation Format: Marc Nater, Giulia Lucchiari, Michael Brügger, Tomas Valenta, Virginia Cecconi, Geo Forni, Stephan Benke, Maries van den Broek. CD1d-restricted iNKT cells promote liver metastasis from colon 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 1386.
Abstract 1387: SETD2 loss promotes macrophage infiltration and metastasis in RCC
Authors: Arner Emily N. , Wolf Melissa M. , Vlach Logan , Rathmell Jeffrey C. , Rathmell W. Kimryn
Published: March 2024

Across human cancer, tumors that are high grade, poorly differentiated, and have undergone epithelial-to-mesenchymal transition (EMT) carry a worse prognosis with a high likelihood of metastasizing to distant organs. Despite significant research efforts, metastatic cancers and relapse remain the primary cause of cancer related deaths. In renal cell carcinoma (RCC), metastasis is present in one-third of patients at the time of diagnosis, and one-third of patients who initially present with locally advanced disease eventually develop local or distant recurrence following resection of the primary tumor, highlighting the need for new RCC therapies and further insight into the metastatic process. Using RCC patient biopsies, we found that patients with more epithelial tumors have a significantly lower risk of recurrence. Additionally, tumors that had a mesenchymal protein signature had significantly increased macrophage infiltration, which was significantly associated with a higher stage at diagnosis and worse overall survival, suggesting that macrophages may play an important role in EMT and metastasis in ccRCC. Whole exome sequencing of these patient samples revealed that mutational loss of SETD2, a histone methyltransferase that is lost in about 20% of RCC patients, correlated strongly with increased macrophage infiltration within the primary tumor and was necessary for the correlation between macrophages and EMT. SETD2 loss in syngeneic kidney cancer mouse models confirmed that SETD2 loss within tumor cells resulted in increased EMT, macrophages within the primary tumor microenvironment (TME), and spontaneous metastasis, further supporting the role of macrophages in promoting metastasis in the absence of SETD2. Further characterization of the gene signatures and immune cell infiltrate in RCC will lead to a better understanding of metastasis in an oncogene- and tissue-specific manner and thus lead to more efficacious therapies for metastatic patients.

Citation Format: Emily N. Arner, Melissa M. Wolf, Logan Vlach, Jeffrey C. Rathmell, W. Kimryn Rathmell. SETD2 loss promotes macrophage infiltration and metastasis in RCC [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 1387.
Abstract 1389: Understanding the differential role of an IL-6 family cytokine in varying subtypes of breast cancer patients for novel therapeutic intervention
Authors: Wolf Cody L. , Tawara Ken , Jorcyk Cheryl L.
Published: March 2024

In the United States, it is estimated that over 295,000 women will be diagnosed with breast cancer, and over 43,000 breast cancer deaths in 2023. Mortality in breast cancer is due in large part due to distant metastasis to organs such as the bone, lung, liver, and brain. Our laboratory has identified that the IL-6-family cytokine oncostatin M (OSM) plays a crucial role in initiating metastasis by secreting pro-metastatic molecules such as VEGF, LOXL2 and IL-6, and increasing circulating tumor cell numbers and metastases to bone and lung in vivo. More recent work within our lab has identified unique roles for OSM in varying subtypes of breast cancer patients, particularly between estrogen receptor positive (ER+) and ER- patients. It is well known that patients with ER- breast cancer, specifically triple negative breast cancer (TNBC; ER- PR- HER2-), have limited therapeutic options. However, the divergent role OSM plays in ER+ and ER- breast cancer and the mechanism by which this occurs has yet to be fully understood. Thus, we aimed to fully elucidate the difference between OSM in ER+ versus ER- breast cancer. RNA sequencing analysis on OSM-treated human ER+ cells and ER- TNBC cells discovered significant differences in gene expression between both cell lines upon treatment with OSM, including alteration in DNA-replication and cell cycle pathway genes. Afterwards to evaluate the correlation between OSM and DNA synthesis and this role on metastasis, we evaluated the role of a novel anti-OSM therapeutic and its correlation with decreased metastasis. This research aims to elucidate the unique role OSM plays in ER+ and ER- breast cancer and will provide a molecular understanding of how this occurs and will provide detailed information on which patients will benefit most from anti-OSM therapeutics.

Citation Format: Cody L. Wolf, Ken Tawara, Cheryl L. Jorcyk. Understanding the differential role of an IL-6 family cytokine in varying subtypes of breast cancer patients for novel therapeutic intervention [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 1389.
Abstract 144: Sex bias in neuroblastoma: Worse outcomes for female patients are associated with dysregulation of DLK1 and H19
Authors: Raleigh Mariya , Patel Vishwa , Benhamou Lorraine R. , Shen Xin , Sheeter Dennis A. , Espinosa Erika C. , Badi Niharika Reddy , Calvert Chase , Bourne Garrett , Powers John T.
Published: March 2024

Neuroblastoma (NB) is a devastating neural crest-derived tumor primarily affects children under five. Forty percent of NB patients are classified as high-risk (HR), and less than half of them survive. HR disease is split into two equally sized groups: one exhibiting genetic amplification of the MYCN proto-oncogene (MA), and another where MYCN is non-amplified (MNon). Overall survival (OS) studies are often either poorly annotated or focused on outcomes across the entire patient group, which may mask differential outcomes or biases among NB subgroups. Here we report our assessment of OS among several genetic, histopathological, and sex-based subgroups in three different well annotated NB patient datasets. Our analysis reveals a previously unseen sex-bias in HR MNon patients, where female patients have significantly worse OS than males. This disparity carries into stage 4s NB, defined by high survival in infants despite the presence of broadly disseminated disease. In stage 4s patients across two studies, we observe no deaths among males (n=66) and 6 among females (n=48), again marking significantly worse OS in females. Expression analysis comparing male and female HR MNon patients identifies DLK1 and H19, two imprinted genes implicated in multiple malignancies, as frequently overexpressed in females. A similar pattern is seen in RNA seq analysis of human MA (n=4) and MNon (n=4) cell lines. In addition, both DLK1 and H19 are associated with worse prognosis in female but not male patients. We also report that depletion of either gene results in reduced growth in vitro. Moreover, the NB-related ATRX tumor suppressor has been shown to inhibit H19 via insertion of histone variant H3.3. We observe an inverse correlation between H3.3B and H19 expression in female HR MNon patients. Further, H19 levels increase upon ATRX depletion in human NB cells, suggesting that ATRX indeed regulates H19 in NB. These findings provide the first evidence of female-biased outcomes in NB and suggest functional involvement of DLK1, H19 and ATRX.

Citation Format: Mariya Raleigh, Vishwa Patel, Lorraine R. Benhamou, Xin Shen, Dennis A. Sheeter, Erika C. Espinosa, Niharika Reddy Badi, Chase Calvert, Garrett Bourne, John T. Powers. Sex bias in neuroblastoma: Worse outcomes for female patients are associated with dysregulation of DLK1 and H19 [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 144.
Abstract 1458: Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness
Authors: Mosna Jimena , Garde Federico , Stinson Marcelo , Pastore Candela , Sanchis Pablo , Carcagno Abel
Published: March 2024

Pancreatic ductal adenocarcinoma (PDAC) represents a very aggressive type of pancreatic cancer (9% survival rate at 5 years). PDX1 is an important transcription factor for embryonic development of the pancreas, endocrine lineage differentiation and the maintenance of mature beta cells. Notably, in PDAC patients, the expression of PDX1 is downregulated in tumor cells compared to adjacent non-tumoral tissue. Interestingly, increasing tumor PDX1 expression has been shown to enhance the overall survival rate in PDAC patients. Therefore, our aim was to analyze the role of PDX1 on tumor aggressiveness of PDAC cells. To induce PDX1 expression, PANC1 cells were treated with BRD7552 (a PDX1 inducer) for 9 days. Overexpression of PDX1 was confirmed by Western Blot analysis and no cytotoxic effects were observed by MTT or Trypan Blue exclusion assays on treated cells compared to control. Wound healing and transwell migration assays showed a significant reduction in the migration rate compared to the control group. A higher proportion of cells in G1 phase was observed in treated cells compared to control by propidium iodide staining followed by flow cytometry assay. Furthermore, the cell confluence assay showed a significant reduction in proliferation rate in treated cells compared to control, but no differences were observed on the proportion of KI67+ and PH3+ cells by immunostaining. To assess the in ovo effects of BRD7552, treated PANC1 cells were implanted onto the chorioallantoic membrane of chick embryos and tumor growth was measured at different stages observing a significant reduction in tumor growth in PANC1 cells compared to control between days 3 and 8 post implantation. No significant differences in morphology or color were observed between treated and control implanted cells. Hematoxylin-eosin staining showed reduced invasion of treated cells into the chorioallantoic membrane compared to control. In conclusion, the overexpression of PDX1 affects cell cycle, reduces proliferation rate and inhibits migratory potential in vitro. Moreover, in an in ovo model this overexpression diminished tumor growth and invasion in PANC1 cells. In conclusion, the overexpression of PDX1 induced by BRD7552 drives pancreatic ductal adenocarcinoma to a less aggressive tumoral phenotype.

Citation Format: Jimena Mosna, Federico Garde, Marcelo Stinson, Candela Pastore, Pablo Sanchis, Abel Carcagno. Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness [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 1458.
Abstract 1470: Advancing cancer research: A novel PanCancer digital PCR tool for simultaneous detection of multiple hallmark mutations in BRAF and EGFR
Authors: Kappmeier Claudia , Edward Sherina , Hochstein Corinna , Bruske Ellen Inga , Pasquale Francesca Di , Kellner Ronny
Published: March 2024

The paradigm shift towards precision medicine in oncology emphasizes the importance of identifying specific genetic mutations driving cancer. Some hallmark mutations in cancer-associated genes, such as BRAF and EGFR, are important in analyzing many cancer types. With the emergence of digital PCR (dPCR) technology, high-sensitivity and quantitative mutation analysis can be performed, lending valuable support to cancer research. In this work, we introduce a new and innovative dPCR method, the dPCR PanCancer assays (RUO), designed for concurrently detecting multiple hallmark mutations in BRAF and EGFR.

This work includes two novel PanCancer assays for BRAF and EGFR, each tailored to target a spectrum of mutations associated with these genes, facilitating a comprehensive mutation analysis. The dPCR assay for BRAF targets multiple V600 hallmark mutations, and the dPCR assay for EGFR targets various important deletions on exon 19. This also includes a reference gene as a control for PCR efficiency in the duplex reaction. Here, we present our initial data from various sample types, including blood, plasma and FFPE samples. Through a meticulously optimized dPCR setup, we achieved exceptional sensitivity and specificity, enabling the detection of multiple mutations in a single channel at allelic frequencies below 1%.

Both dPCR PanCancer Kits (RUO) have potential use in research for pre-screening samples (e.g., prior to next-generation sequencing) or monitoring cancer cells. They simultaneously assess the mutations, reducing time and costs and saving sample material. Additionally, our technology is adaptable to other cancer-associated genes, where similar assays can potentially be developed. Overall, we have demonstrated that our dPCR PanCancer Kits (RUO) provide a robust, fast and efficient technology to identify critical mutations, ultimately enhancing our understanding of BRAF and EGFR-driven cancers.

The dPCR PanCancer Kit is for research use only. Not for the diagnosis, prevention, or treatment of a disease.

Citation Format: Claudia Kappmeier, Sherina Edward, Corinna Hochstein, Ellen Inga Bruske, Francesca Di Pasquale, Ronny Kellner. Advancing cancer research: A novel PanCancer digital PCR tool for simultaneous detection of multiple hallmark mutations in BRAF and EGFR [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 1470.
Abstract 1472: APOBEC-induced non-coding hotspot mutations near LEPROTL1 contribute to the tumorigenicity of bladder cancer via deregulation of LEPROTL1, DCTN6 and SARAF
Authors: Lone Bilal , Butler Kelly , Dobbins Alexandra , Yesudhas Dhanusha , Chakraborty Arup , Hickman Evan , Boudjadi Salah , Apolo Andrea , Banday Rouf
Published: March 2024

In bladder cancer, APOBEC3 deaminases are major drivers of genomic mutagenesis, with most of these mutations occurring in synonymous or non-coding regions of the genome. The impact of these mutations on tumorigenesis has not been explored. This study aimed to investigate two non-coding hotspot mutations, likely induced by APOBEC3A near LEPROTL1—chr8: 29,952,919 G>A and chr8: 29,952,921 C>T—in bladder cancer. Using dual-luciferase reporter assays, we found that these mutations enhance both promoter and enhancer activities. Employing multi-omics approaches, including RNA-seq, ChiP-seq, and Hi-C, identified LEPROTL1, DCTN6, and SARAF as target genes co-regulated by a common enhancer where these hotspot non-coding mutations manifest. Isogenic cell lines, generated through CRISPR-mediated base editing to introduce these mutations, exhibited upregulation of target genes, and displayed oncogenic characteristics, such as increased cell growth, proliferation, invasiveness, and resistance to cisplatin. Moreover, these mutations exert their effect on cell proliferation by promoting cell cycle progression. These results suggest that recurrent non-coding somatic mutations upstream of LEPROTL1 disrupt the coregulation pattern of the three genes, thereby promoting bladder tumorigenesis.

Citation Format: Bilal Lone, Kelly Butler, Alexandra Dobbins, Dhanusha Yesudhas, Arup Chakraborty, Evan Hickman, Salah Boudjadi, Andrea Apolo, Rouf Banday. APOBEC-induced non-coding hotspot mutations near LEPROTL1 contribute to the tumorigenicity of bladder cancer via deregulation of LEPROTL1, DCTN6 and SARAF [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 1472.
Abstract 149: Modeling patient-specific CHEK2 genetic variants associated with high-risk neuroblastoma
Authors: Xiong Xueting , Cohen-Gogo Sarah , Villani Anita , Shlien Adam , Irwin Meredith S. , Hayes Madeline
Published: March 2024

Background: Neuroblastoma (NB) is the most common extra-cranial solid tumor in children and accounts for approximately 10% of childhood cancer deaths. Recently, next-generation sequencing of patients enrolled in the SickKids Cancer Sequencing (KiCS) program revealed somatic and germline alterations in genes involved in the DNA damage response (DDR). Multiple germline and somatic pathogenic variants in CHEK2 were detected in patients with NB.

Aims and Methods: To better understand potential roles for CHEK2 in NB pathogenesis, we are modeling patient-derived CHEK2 variants in an established transgenic zebrafish model of MYCN-driven NB. Using a CRISPR/Cas9 “knock-in” approach, we have generated stable zebrafish lines that are homozygous and heterozygous for variants of interest with either null, heterozygous, or wild-type tp53 functional status. We aim to assess tumor incidence, growth, and metastasis using direct visualization of disseminated EGFP+ tumor cells, as compared to MYCN over-expression only control fish. Given potential drug sensitivities, we also plan to assess the efficacy of various DDR pathway inhibitors in combination with frontline chemotherapies, which are effective in the treatment of DDR-deficient adult cancers.

Results: Targeting chek2 in a tp53 null background had no significant effect on tumor incidence and growth compared to tp53 mutant control animals (p=0.1570 log-rank test). This contrasts with other patient-specific DDR models including gene targeting brca2 (p=0.0235, log-rank test), atm (p<0.001, log-rank test), and bard1 (p<0.001, log-rank test), potentially highlighting Tp53-dependent roles for Chek2 in NB formation in vivo. Through functional characterization of patient-specific CHEK2 variants in NB progression, our work will provide important preclinical information that will inform future diagnostics and therapeutic strategies for patients with high-risk NB.

Citation Format: Xueting Xiong, Sarah Cohen-Gogo, Anita Villani, Adam Shlien, Meredith S. Irwin, Madeline Hayes. Modeling patient-specific CHEK2 genetic variants associated with high-risk neuroblastoma [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 149.
Abstract 1491: The enhanced formation of neutrophil extracellular traps (NETs) may contribute to the exacerbation of peritoneal metastasis in a diabetic host
Authors: Takahashi Rei , Ohzawa Hideyuki , Kaneko Yuki , Matsumiya Misaki , Futoh Yurie , Tamura Kohei , Takahashi Kazuya , Kimura Yuki , Saito Akira , Miyato Hideyo , Sata Naohiro , Kitayama Joji
Published: March 2024

Background: Diabetes is associated with increased risk of cancer development and progression. Recent studies have shown that neutrophils in diabetic patients are prone to forming neutrophil extracellular traps (NETs) which have been reported to capture circulating cancer cells, leading to the promotion of liver and lung metastasis. This study aims to clarify the causative relationship between NETs and the peritoneal metastasis (PM) in diabetic condition.

Methods: Outcome of the diabetic patients who received curative gastrectomy for gastric cancer from 2010 to 2021 in our institute was comparatively investigated with those without diabetes. Neutrophils were isolated from the bone marrow from C57BL/JHamSlc-ob/ob mouse (type 2 diabetes model) and wild type mouse using Ly6G-based positive immunomagnetic selection. The neutrophils were stimulated with 100nM of PMA for 4 hours and NETs formation was evaluated by the addition of Sytox Green under fluorescent microscope. A mouse gastric cancer cell, YTN16P (5 × 105 cells) was intraperitoneally (IP) injected, the number of metastatic nodules on mesentery was comparatively evaluated two weeks later.

Results: Disease-free survival (p=0.004, HR=1.65) and overall survival (p=0.035, HR=1.46) of 203 diabetic patients were significantly worse those of 975 non-diabetic patients. Neutrophils isolated from ob/ob mice produced markedly increased NETs than that from wild-type mice. NETs produced by neutrophils obtained from the peritoneal cavity of ob/ob mice after IP injection of a 9% casein solution effectively captured many YTN16P in vitro. The concentration of double strand DNA (dsDNA) in plasma in ob/ob mice compared with their counterparts (676ng/mL vs 590ng/mL, p=0.036, n=3). The number of PM in ob/ob mice was significantly more than that in wild-type controls (39.1 vs 15.5, p=0.027, n=10).

Conclusion: Enhanced NETs formation in diabetic conditions may be associated with an increased risk of PM in gastric cancer.

Citation Format: Rei Takahashi, Hideyuki Ohzawa, Yuki Kaneko, Misaki Matsumiya, Yurie Futoh, Kohei Tamura, Kazuya Takahashi, Yuki Kimura, Akira Saito, Hideyo Miyato, Naohiro Sata, Joji Kitayama. The enhanced formation of neutrophil extracellular traps (NETs) may contribute to the exacerbation of peritoneal metastasis in a diabetic host [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 1491.
Abstract 1498: TGFβ signaling in cancer-associated fibroblasts drives a hepatic gp130-dependent pro-metastatic inflammatory program in CMS4 colorectal cancer
Authors: Abudukelimu Subinuer , Harryvan Tom J. , Janson Stef G. , Stouten Imke , Verdegaal Els M. , Hawinkels Lukas JAC
Published: March 2024

Background: Colorectal cancer (CRC) is characterized by a metastatic pattern that shows tropism towards the liver which is the major cause of CRC-related deaths. The current CRC classification, Consensus Molecular Subtypes (CMS), suggests an association between transforming growth factor (TGF)-β signaling, cancer-associated fibroblasts (CAFs), and the risk of liver metastasis. However, the key downstream effectors of TGFβ signaling in CAFs and their role in hepatic metastases remain poorly understood.

Methods: The aim of this study was to investigate the TGF-β/CAF dependent cross talk with the hepatocytes in pre-metastatic niche formation. CAFs from 18 primary CRC tissues and 7 liver-metastasized CRC were stimulated with TGFβ1 and qPCR arrays/ELISAs were performed to identify differentially expressed genes. Results were cross-referenced with RNAseq expression data of CMS-classified CRC and in fresh samples from CRC patients. To investigate CAF-liver crosstalk hepatocytes were stimulated with the TGFβ primed CAF-derived cytokines and the effect on pro-inflammatory gene expression and neutrophil migration was determined. Subsequently, blockade of these pathways via chemical inhibition and CRISPR/Cas9 genetic ablation was performed to study the molecular mechanism involved in the induction of this hepatic pro-inflammatory program.

Results: TGFβ signaling in primary CRC-derived CAFs leads to increased expression of different IL-6 cytokine family members, which was also reflected in human CRC samples. CAF-derived IL6 family members induce upregulation of myeloid chemoattractants, including SAA1, in hepatocytes and increased neutrophil-to-hepatocyte migration in vitro. Chemical blockade and genetic ablation of the IL-6 family cytokine signaling pathway showed the critical role of the gp130 co-receptor in the regulation of this inflammatory response in hepatocytes and the potential formation of a premetastatic niche.

Conclusion: TGFβ signaling in CAFs actively contributes to the formation of a neutrophil-dependent, pre-metastatic hepatic niche, and this mechanism might play a role in CMS4 subtype CRC.

Citation Format: Subinuer Abudukelimu, Tom J. Harryvan, Stef G. Janson, Imke Stouten, Els M. Verdegaal, Lukas JAC Hawinkels. TGFβ signaling in cancer-associated fibroblasts drives a hepatic gp130-dependent pro-metastatic inflammatory program in CMS4 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 1498.
Abstract 152: Exploring the impact of heat shock protein 90 on the antitumor efficacy and stemness in medulloblastoma cells
Authors: Chen Shu-Mei , Huang Hsieng-Yun , Wang Chien-Kai , Yang Yi-Yuan , Leu Sy-Jye , Li Ying-Ying
Published: March 2024

Background: Medulloblastoma (MB) is the most common children’s malignant brain tumor. Although MB can be treated with conventional chemotherapies, chemoresistance has been frequently developed and thereby hampers their therapeutic effectiveness. The preliminary reports of our research showed that some heat shock proteins have higher levels in MB cells. Among these heat shock proteins, HSP70 and HSP90 expressions are particularly high in MB CD133+ cancer stem cells (CSCs), which arouses our attention and interest. Numerous studies demonstrated that heat shock proteins are prominent in some chemotherapy-resistant cancer cells and CSCs. As known HSP90 and its client proteins are required for the maintenance of self-renewal/stemness and regulation of cell cycle, synergistic killing of cancer cells by simultaneous inhibition of HSP90 expression and activity may be a considerable good therapeutic strategy for treating MB. The purpose of this study is to verify the regulatory function of HSP90 in chemoresistance and stemness of MB.

Materials and Methods: The levels of HSP90, its client proteins (P13K/pAKT, Erk, NFκB, and c-Myc), and stemness makers were detected using immunoblotting and immunoprecipitation after the treatment of HSP90 inhibitor (17-AAG) or knockdown of HSP90 by siRNA in MB and MB CSCs. The antitumor effects of HSPs inhibitors or knockdown of HSPs on these cells were evaluated by MTT assay, Annexin V/PI analysis and cell cycle analysis.

Results: Our findings revealed elevated expression of HSP90 and its target client proteins in MB cells compared to normal astrocytes. Silencing or inhibiting HSP90 expression induced cell cycle arrest in MB cells. Notably, CD133+ MB CSCs exhibited higher HSP90 levels than parental MB cells. Inhibition of HSP90 led to reduced sphere formation and expression of stemness markers in CD133+ MB CSCs. Treatment with 17-AAG demonstrated antitumor activity in CD133+ MB CSCs, and a synergistic effect was observed when combining conventional chemotherapeutic agents with 17-AAG in MB cells

Conclusion: These results illustrated that inhibition of HSP90 could eliminate CSCs, and thereby overcome resistance to chemotherapeutic agents in MB. Chemotherapies in combination with HSP90 blockade may be an emerging MB therapy in the future.

Citation Format: Shu-Mei Chen, Hsieng-Yun Huang, Chien-Kai Wang, Yi-Yuan Yang, Sy-Jye Leu, Ying-Ying Li. Exploring the impact of heat shock protein 90 on the antitumor efficacy and stemness in medulloblastoma 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 152.
Abstract 1521: Chromatin remodeler ACTL6A in CD8+ T cells promotes antitumor immunity
Authors: Wu Bogang , Thant Win , Bitman Elena , Liu Ting , Ellisen Leif W.
Published: March 2024

Chromatin remodeling plays a pivotal role in dictating epigenetic and transcriptomic regulation governing cytotoxic T lymphocyte differentiation and effector function. However, the regulatory mechanisms underlying chromatin remodeler assembly and actions remains unclear. Actin-like gene family members have been implicated in the organization of chromatin landscapes, however their role in T cell functional regulation remains unknown. We identified Actin-like protein 6A/Actl6a as the most upregulated gene within actin-like family members upon CD8+ T cell activation across multiple species. Additionally, ACTL6A is highly expressed in human tumor-infiltrating CD8+ mitotic tissue-resident memory T cells, suggesting a functional importance of ACTL6A in this subset. Deletion of mouse Actl6a in mature T lymphocytes impairs immune responses to transplanted syngeneic mouse tumors. T cell-specific Actl6a deletion mice exhibit a reduced effector memory population and attenuated cytotoxicity in their tumor infiltrating CD8+ T cells. Heterozygous deletion of Actl6a displays an intermediate reduction of effector memory population and cytotoxicity between that of wildtype and homozygous knockout, indicating that ACTL6A acts in a dose-dependent manner to promote CD8+ effector cell fate. T cell adoptive transfer further corroborates a CD8+ T cell-intrinsic role of Actl6a on antitumor adaptive immunity. Currently, we are investigating the chromatin landscape and transcriptomic changes upon loss of Actl6a using ATAC-seq and RNA-seq. Furthermore, the dose dependency of Aclt6a on CD8+ T cell effector function suggests that pharmacological or genetic approaches to increase Actl6a expression in CD8+ T cells may have the potential to boost immune based anticancer therapies including CAR-T therapies.

Citation Format: Bogang Wu, Win Thant, Elena Bitman, Ting Liu, Leif W. Ellisen. Chromatin remodeler ACTL6A in CD8+ T cells promotes 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 1521.
Abstract 1537: CD63 acts as a key modulator of tumor progression and innate immune evasion in non-small cell lung cancer
Authors: Lerbs Tristan , Cui Lu , De Souza Cristabelle , Buettner Reinhard , Wernig Gerlinde
Published: March 2024

Introduction: Lung cancer is a major reason for cancer-related mortality worldwide, causing more than two million cancer deaths per year. Over the last decade, new therapies have allowed long-term survival for patients even in advanced stages. However, only a minority of patients benefits from these therapies and patients often grow resistant over time. An important mechanism that contributes to cancer growth and the development of therapy resistances is the cross-talk between cancer cells and their tissue microenvironment, in particular fibroblasts and macrophages. In this context, we explored whether the tetraspanin CD63 - which is commonly found on intra- and extracellular membranes - stimulates cancer growth and mediates pro-tumorigenic functions of the tissue microenvironment in NSCLC.

Methods: We analyzed patient tissue via immunohistochemistry and in-situ-hybridization. We deleted CD63 in cancer cell lines (A549, H810, LC19) and primary fibroblasts via CRISPR-Cas9. To assess phagocytosis, we used in vitro EdU assays and adaptive transfer models in vivo, and identified changes in cell signaling via mass spectrometry. We used an in vitro phagocytosis assay to determine the effect of CD63 on cancer cell phagocytosis through macrophages and analyzed the expression of M1 and M2-associated surface markers through flow cytometry upon co-culturing PBMC with cancer cells over one week.

Results: In patient sections, cancer tissue did not only express more CD63 on the gene expression and protein level but a stronger CD63 expression also correlated with a denser macrophage infiltrate. Looking for pro-tumorigenic functions, deleting CD63 in three cancer cell lines reduced cancer cell growth both in vitro and in an adaptive transfer model in vivo. Additionally, deleting CD63 impaired the pro-tumorigenic focal adhesion signaling in primary fibroblasts. Concerning the interaction between cancer cells and macrophages, deleting CD63 reduced the macrophage-mediated phagocytosis of cancer cells and co-culturing primary monocytes with CD63-deficient lung cancer cells induced the expression of M1-associated surface markers on the PBMC.

Conclusion: In conclusion, these results demonstrate that the tetraspanin CD63 drives NSCLC growth by stimulating cancer cell proliferation - in part by interfering with focal adhesion signaling - and facilitating immune evasion by preventing cancer cell phagocytosis through macrophages and inducing an immunosuppressive phenotype in macrophages.

Citation Format: Tristan Lerbs, Lu Cui, Cristabelle De Souza, Reinhard Buettner, Gerlinde Wernig. CD63 acts as a key modulator of tumor progression and innate immune evasion in non-small cell lung 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 1537.