bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1356: The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation
Authors: Dharmadhikari Bhushan , Zharkova Olga , Thakkar Dipti , Tirado-Magallanes Roberto , Paszkiewicz Konrad , Ingram Piers J. , Boyd-Kirkup Jerome D.
Published: March 2024

VISTA, a predominantly myeloid immune checkpoint, promotes an immunosuppressive tumor microenvironment and has been associated with resistance to approved checkpoint therapies. While blockade of VISTA with antibodies has shown anti-tumor efficacy in preclinical models, the biological mechanisms that underlie this effect remain poorly understood. To investigate the mechanistic basis of anti-tumor responses, we performed single-cell RNA sequencing of tumor infiltrating leukocytes from the cell-derived murine colon carcinoma xenograft model, CT26, which is known to be sensitive to VISTA blockade. We used HMBD-002, a uniquely blocking but non-depleting clinical-stage IgG4 anti-VISTA antibody, to identify the key immune cells and pathways impacted by VISTA blockade in the absence of Fc-mediated cell depletion. The translatability of these findings was validated in a human PBMC mixed leukocyte reaction assay. Subsequently, targeted depletion or inhibition of the identified immune populations and pathways was performed to confirm their contribution to the anti-tumor efficacy of HMBD-002.The blockade of VISTA by HMBD-002 significantly increased CD8+ T cells in the CT26 tumors, promoting an activated cytotoxic Tc1 program without inducing exhaustion. Concurrently, a substantial increase in proinflammatory M1 tumor-associated macrophages (TAMs) was noted within treated tumors, while other immune cell populations remained unaffected. Among the signaling pathways, the type-I interferon (IFN) response exhibited a significant upregulation, particularly in TAMs, indicating a type-I IFN-driven M1 reprogramming by HMBD-002. Validation experiments further demonstrated that the depletion of TAMs or CD8+ T cells, but not CD4+ T cells, abrogated the efficacy of HMBD-002. Moreover, blocking the type-I IFN signaling effectively impeded HMBD-002-induced TAM reprogramming and subsequent anti-tumor efficacy, thus confirming the pivotal roles of CD8+ T cells, TAMs, and type-I IFN signaling in driving the anti-tumor response to VISTA blockade. In conclusion, VISTA blockade with HMBD-002 promotes an anti-tumor response by type-I IFN mediated reprogramming of TAMs and activation of cytotoxic CD8+ T cells.

Citation Format: Bhushan Dharmadhikari, Olga Zharkova, Dipti Thakkar, Roberto Tirado-Magallanes, Konrad Paszkiewicz, Piers J. Ingram, Jerome D. Boyd-Kirkup. The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation [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 1356.
Abstract 1371: SLAMF9 promotes tumor progression and immunotherapy resistance by reshaping the tumor microenvironment
Authors: Fan Tao , Xiao Chu , Deng Ziqin , Cai Wenpeng , Tian He , Wang Liyu , Li Chunxiang , He Jie
Published: March 2024

Tumor prognosis and immunotherapy efficacy coincides with the infiltration ratio and functional status of immune cells in the tumor microenvironment (TME). Signaling lymphocytes activating molecule factors (SLAMFs) has been reported to play a key role in regulating tumor immunity. Herein, using multi cohorts, in vitro and in vivo experiments, we showed that SLAMF9 worsens tumor and suppresses immune therapy responses by remodeling TME. A pan-cancer analysis showed that SLAMF9 had relatively high transcriptional levels within tumor tissues compared with normal tissues across most cancer types. High SLAMF9 expression is associated with poor prognosis and anti-PD-L1 therapy resistance in advanced clear cell renal cell carcinoma cohorts and metastatic urothelial cancer cohorts. SLAMF9 expression was markedly upregulated in anti-CTLA-4-resistant murine B16 melanoma compared with parental tumors. In particular, SLAMF9 expression was generally negatively correlated with CD8+T cell infiltration score in 12159 patients of 40 cancer types even using different immune cell infiltrating algorithms. Although in vitro experiments have shown that SLAMF9 knockdown has almost no effect on the malignant phenotype of tumor cells, subcutaneous melanoma and lung adenocarcinoma with SLAMF9 deficiency grew slowly, which featured with increased infiltration of CD8+T and PD-1+cells. More importantly, SLAMF9-knockdown significantly increases the sensitivity of subcutaneous melanoma to anti-CTLA-4 inhibitor or anti-PD-L1 antibodies and improved the survival benefits of tumor-bearing mice. This work highlights SLAMF9 as a new target in reshaping the tumor immune microenvironment by controlling the functional status of CD+8 T cells and to improve the efficacy of immunotherapy.

Citation Format: Tao Fan, Chu Xiao, Ziqin Deng, Wenpeng Cai, He Tian, Liyu Wang, Chunxiang Li, Jie He. SLAMF9 promotes tumor progression and immunotherapy resistance by reshaping the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1371.
Abstract 1391: Therapeutic salmonella induces long-term protective trained immunity in NK cells against metastasis
Authors: Rong Li , Huang Jiandong
Published: March 2024

Immune memory is a trait commonly associated with adaptive immunity. Nevertheless, it has been observed that activation of the innate immune system can also result in enhanced responsiveness to subsequent triggers, a form of innate immune memory known as trained immunity. This phenomenon has been primarily explored in the context of host defense against infectious disease, where myeloid cells, including monocytes, macrophages, and their precursor cells, are conditioned by specific stimuli such as β-glucan, LPS, or the bacillus Calmette-Guιrin (BCG) to boost protection against microbial pathogens. However, the induction of trained immunity in innate lymphocytes, particularly through these known or yet-to-be-discovered stimuli and its explicit contribution to tumor immunity, remain insufficiently characterized. Metastasis accounts for 90% of cancer-related deaths, and no effective clinical therapies are currently available to halt the metastatic cascade. Based on multiple cancer metastasis mouse models and immunological analysis, we found that a single dose of therapeutic Salmonella YB1, an engineered oxygen-sensitive strain, provides extensive and persistent protection against metastasis by inducing protective trained immunity in NK cells, consistently observed irrespective of the mouse genetic background or cancer cell lines. Following inflammatory resolution, salmonella YB1-trained NK cells exhibit an enhanced capacity to release IFN-γ upon secondary stimulation and increased cytotoxicity towards cancer cells, which is associated with epigenetic changes. Further investigation using single-cell transcriptomic profiling indicates that the formation of long-term anti-metastatic trained immunity in NK cells by Salmonella YB1 is dependent on IL12 and IL18. Additionally, Salmonella YB1-trained NK cells outperform common immune checkpoint therapies like PD1 blockade and TIGIT blockade in suppressing metastasis. In summary, our results revealed a novel anti-metastatic function of trained NK cells induced by therapeutic Salmonella, suggesting the induction of trained immunity in NK cells as a potential anti-tumor strategy.

Citation Format: Li Rong, Jiandong Huang. Therapeutic salmonella induces long-term protective trained immunity in NK cells against 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 1391.
Abstract 147: Identification of cell sub-populations in Neuroblastoma cell lines with single-cell-sequencing analysis
Authors: Tomanelli Michele , Ceresa Davide , Bajetto Adriana , Modesto Paola , Malatesta Paolo , Florio Tullio , Pagano Aldo
Published: March 2024

Neuroblastoma is the most frequent extracranial tumor in neonatal patients. Engineering SKNBE2 Myc-N amplified Neuroblastoma cell lines we generated individual clones expressing NDM29 non-coding-RNA at different levels. Using these cell models we documented a specific role of NDM29 in tumor cell differentiation leading to the identification of novel genes to be pharmacologically targeted for an efficient control of tumor malignancy (CA9 and MCM2 genes). Further studies taking advantage of the same models suggested the possible co-existence of different cell sub-populations in the apparently homogeneous cell culture with possible different roles in tumor maintenance and growth. Here we test this hypothesis by performing single-cell sequencing analysis and identified different sub-populations on the base of their gene expression patterns. The characterization of their specific profiles of membrane markers allowed an efficient separation of the individual populations by FACS analysis. Our preliminary results show the co-existance of cells with a marked neural phenotype with other two populations one VEGF secreting and the other VEGF recipient/responder. These preliminary findings suggest further detailed investigations of possible paracrine loops that might be at the base of tumor cell nodule growth and maintenance.

Citation Format: Michele Tomanelli, Davide Ceresa, Adriana Bajetto, Paola Modesto, Paolo Malatesta, Tullio Florio, Aldo Pagano. Identification of cell sub-populations in Neuroblastoma cell lines with single-cell-sequencing analysis [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 147.
Abstract 1482: FKBP52 drives diverse HCC phenotypes through context-dependent targeting of p53
Authors: Wang Xing , Wu Jingfeng , Tang Chengyan , Li Xiang , Zhu Li , Liu Yaoyu
Published: March 2024

p53 is notably the most frequently mutated protein in hepatocellular carcinoma (HCC), one of the major causes of cancer-related fatalities globally. These mutations often result in some of the most adverse outcomes in HCC, making p53 a prime target for therapeutic intervention. The concept of “hyperstability”of p53 is essential in understanding its gain-of-function and dominant-negative effects in tumorigenesis. However, the mechanisms driving this hyperstability are not completely understood. In this study, we have identified FKBP52, a member of the FK506-binding protein family, as a crucial factor in stabilizing and accumulating p53, irrespective of the type of mutation. Furthermore, our study suggests that FKBP52 could be a valuable biomarker for predicting HCC outcomes. We observed a notable link between FKBP52 levels and the histological differentiation in HCC samples. To corroborate these observations, we performed a series of in vitro and in vivo experiments using liver cancer models. These experiments highlighted the dual nature of FKBP52’s impact on hepatocytes, which varies according to cell type. Given its connection to HCC severity, FKBP52 seems to contribute negatively to the disease’s progression. The insights gained from this study on how FKBP52 influences p53 stabilization are critical. We suggest that targeting the processes upstream of FKBP52 could lead to more effective HCC treatments. These findings emphasize the importance of further investigating this pathway, potentially paving the way for new strategies in liver cancer therapy.

Citation Format: Xing Wang, Jingfeng Wu, Chengyan Tang, Xiang Li, Li Zhu, Yaoyu Liu. FKBP52 drives diverse HCC phenotypes through context-dependent targeting of p53 [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 1482.
Abstract 1527: Exploring the role of tertiary lymphoid structure in the tumor microenvironment of cholangiocarcinoma
Authors: Chung Shin-Yi , Chen Ming-Huang , Yeh Yi-Chen , Chang Yu-Chan , Wang Yu-Cha
Published: March 2024

Cholangiocarcinoma is the second most common hepatobiliary malignant tumor besides liver cancer, grows from bile duct epithelial cells. However, surgery is the only hope for most patients with cholangiocarcinoma, but the effectiveness of treatment is not outstanding. Even though other methods of treatment, such as chemotherapy and radiation therapy, have limited effects. The immunotherapy and precision treatment have become the latest expectations of patients. Here, we analyzed the TCGA and GEO database to classify the cold and hot tumor using hierarchical clustering. The MCP counter results demonstrated that two genes, MS4A1 and CD79A, could identify the hot and cold tumor subgroups after TCGA data training and similar results show in GEO database. We also performed the tissue array for 105 cholangiocarcinoma tissue samples and found patients with high expression levels of MS4A1 or CD79A have better disease free survival (DFS) and progression free survival (PFS) (p value =0.0131 and 0.0017). In addition, we tested the tertiary lymphoid structures (TLS) signatures in tissue array sections, which is associated with the present of MS4A1 and CD79A. The results shows that patients who received the immunotherapy with TLS signatures have longer overall survival (OS) and PFS (p value =0.017 and 0.006). Collectively, B cell lineages or TLS could be an ideal biomarker to lead the clinical treatment and wish can broader applications in other diseases.

Citation Format: Shin-Yi Chung, Ming-Huang Chen, Yi-Chen Yeh, Yu-Chan Chang, Yu-Cha Wang. Exploring the role of tertiary lymphoid structure in the tumor microenvironment of cholangiocarcinoma [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 1527.
Abstract 1532: Improved reproducibility in multiplex immunofluorescence analysis of the tumor microenvironment using a novel quenching solution
Authors: Levin Matt , Heath Jack , Schrank-Hacker April , Dunn Shelby , Vu Minh-Chau , Hamza Imane , Schwartz David
Published: March 2024

Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used in histopathology because the fixed tissues are stable and easy to store. In the context of immunofluorescence detection, however, endogenous autofluorescence of FFPE tissues can significantly reduce or mask immunofluorescent signals. The autofluorescence of FFPE tissues is caused by a variety of factors, including the presence of endogenous fluorophores, tissue fixation, and processing. While there are several methods that can be used to quench autofluorescence in FFPE tissues, chemical quenchers, which absorb or scatter light, are most frequently used for multiplex IF. Examples of chemical quenchers commonly used in manual multiplex staining include Sudan Black B and TrueBlack; however, these are not chemically compatible for use on automated platforms such as the Leica Biosystems BOND RX. Due to the need for increased throughput and decreased hands-on time, automation has risen in popularity versus manual staining, leading to a need for a versatile quenching solution compatible with automation. Here we demonstrate that NovaPlex Quenching Solution (NPQS), a novel proprietary quenching agent, reduces the background caused by autofluorescence and enhances the signal of the Cell IDx UltraPlex multiplex immunofluorescent (mxIF) assay across tissue types. Qualitative studies showed a decrease in autofluorescent signal in FFPE mouse spleen stained with antibodies against B220/Foxp3/CD3e, suggesting the utility of NPQS in mouse models of cancer and immunity. Similarly, nonspecific background was decreased across FFPE human tissue types, including lung adenocarcinoma stained for CD8/Foxp3/CD3e/CD4 and tonsil stained for CD8/Foxp3/PD-1/CD4. Further statistical studies in FFPE human tonsil tissue stained with CD8/Foxp3/PD-1/CD4 showed there was no significant decrease in dye intensity across all four markers and channels, resulting in a significant increase in signal-to-noise ratio for both CD8 at 490nm and PD-1 at 650nm (p<0.05). These experiments using Cell IDx/LBS UltraPlex mxIF technology demonstrate an effective method to improve the signal-to-noise ratios of fluorophores without reducing signal intensity from any of the target proteins in these parallel multiplex immunofluorescent assays.

Citation Format: Matt Levin, Jack Heath, April Schrank-Hacker, Shelby Dunn, Minh-Chau Vu, Imane Hamza, David Schwartz. Improved reproducibility in multiplex immunofluorescence analysis of the tumor microenvironment using a novel quenching solution [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 1532.
Abstract 1559: IKZF1 plays a crucial role in the spread of melanoma
Authors: Oh Jongwook , Lee Dong Ki , Lee Soyeon , Park Hyun Woo , Gee Heon Yung
Published: March 2024

Melanoma is one of the most fatal type of skin cancer. If melanoma has spread to distant sites, it is refractory to existing therapies with the 5-year survival rate declining to 17%. Metastasis remains as the main barrier to successful therapy, persisting as the main cause of cancer-related death. Thus, understanding metastasis mechanism is vital.

First, we confirmed that IKZF1 gene was more increased in circulating tumor cells than primary tumor cells. Using GFP+ B16F10, we generated an orthotopic melanoma mouse model and isolated GFP+ tumor cells of primary footpad, blood, and lung. In single cell RNA sequencing, IKZF1, IRF8, NFE2 genes were more upregulated in circulating tumor cells than primary tumor cells. We also found these correlations in human melanoma cell line orthotopic mouse model and Braf/Pten mouse model.

Next, we conduct intervention study to find causality in IKZF1 and metastasis. IKZF1 KO led to fewer CTCs and IKZF1 inhibitor, lenalidomide, reduced metastasis in mice.

Collectively, we demonstrate that IKZF1 could contribute the formation of CTC and IKZF1 could be a target for metastasis treatment.

Citation Format: Jongwook Oh, Dong Ki Lee, Soyeon Lee, Hyun Woo Park, Heon Yung Gee. IKZF1 plays a crucial role in the spread of 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 1559.
Abstract 1571: Upregulation of phosphoglyceric acid mutase (PGAM)-1 is related to lymph node metastasis in gastric cancer
Authors: Wang Helei , Liu Zhicheng , Wang Lei , Wang Zitian , Jamieson Christina , Cacalano Nicholas A.
Published: March 2024

Gastric cancer (GC) is the most common malignancy of the digestive system, causing over 750,000 deaths worldwide. Gastric cancer mortality is largely due to its high level of invasiveness and metastasis to distal organs. Thus, it is an urgent priority to identify mechanisms underlying GC metastasis to improve cancer diagnosis develop of effective biological targeting agents. In this study, we demonstrate that phosphoglyceric acid mutase-1 (PGAM1) was preferentially over-expressed in lymph node metastasis (LNM) tissues of GC patients and correlated with poor prognosis. PGAM1 knock down in two human GC cell lines resulted in impaired cell proliferation and increased cell cycle arrest. Further, PGAM knockdown inhibited migration of both GC cell lines, which correlated with decreased expression of MMP2, MMP9 and ICAM-1. In a murine xenograft model, GC cells with a PGAM1 knockdown displayed decreased growth capacity in vivo. Western blot analysis showed that PGAM1 knockdown in SGC7901 and BGC823 decreased the phosphorylation of Src, FAK and Paxillin relative to the parental cell lines. These results indicate that PGAM1 may play an important role in promoting GC lymph node metastasis by driving cell proliferation and activating cytoskeletal reorganization and cell migration through the regulating Src/FAK/Paxillin pathway.

Citation Format: Helei Wang, Zhicheng Liu, Lei Wang, Zitian Wang, Christina Jamieson, Nicholas A. Cacalano. Upregulation of phosphoglyceric acid mutase (PGAM)-1 is related to lymph node metastasis in gastric 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 1571.
Abstract 1601: The role of complement in pancreatic cancer cachexia
Authors: D'Lugos Andrew C. , Callaway Chandler , Judge Sarah M. , Judge Andrew R.
Published: March 2024

Cancer cachexia is a multifactorial condition characterized by skeletal muscle atrophy and dysfunction that impairs longevity and quality of life for the vast majority of cancer patients. Recently, we discovered through unbiased proteomic analyses that the complement system is activated in the skeletal muscle of cachectic pancreatic cancer patients. Therefore, the purpose of this study was to investigate the role of complement in pancreatic cancer cachexia. To accomplish this, we obtained genetically modified, C57Bl6/J congenic mice lacking the central regulator of complement, C3 (C3−/− mice). Wild-type (C57Bl6/J, WT, n=12) and C3−/− mice (n=11) (male, 12 weeks old) received orthotopic injections of mouse pancreatic cancer cells (KPC) into the pancreas, and as non-cancer controls, WT (n=8) and C3−/− (n=4) mice received injections of PBS into the pancreas. Mice were monitored daily and euthanized at IACUC-mandated tumor endpoint (body condition score ≤ 2; range: 14-15 days). In-vivo diaphragm function was assessed immediately prior to euthanasia via M-mode ultrasound. Skeletal muscles (soleus, SOL; tibialis anterior, TA; diaphragm, DIA) were harvested and ex-vivo contractile function was assessed on strips of costal diaphragm. Diaphragmatic excursion and respiratory rate were reduced with KPC tumor burden in WT mice, but preserved in C3−/− mice. Deletion of C3 did not influence the KPC-induced reduction of body mass or KPC tumor growth. Atrophy of limb muscle mass (SOL, TA) and fiber size (TA) during KPC tumor burden was attenuated in C3−/− mice. Maximal specific force of DIA was greater in C3−/− versus WT KPC-bearing mice. KPC tumor burden increased collagen content in the DIA of WT mice, but not C3−/− mice. Diaphragm fiber size was reduced in WT KPC-bearing mice, but preserved in C3−/− mice. KPC tumor burden induced significant increases in immune cell (CD45+) infiltration and fibroadipogenic progenitor cell (PDGFRa+) abundance in the DIA of WT tumor-bearing mice, but to a lesser extent in C3−/− mice. These findings suggest complement activation is causative in pancreatic cancer-induced skeletal muscle wasting and dysfunction. Inhibition of complement during cancer cachexia may possess therapeutic potential for the preservation of skeletal muscle mass and function.

Citation Format: Andrew C. D'Lugos, Chandler Callaway, Sarah M. Judge, Andrew R. Judge. The role of complement in pancreatic cancer cachexia [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 1601.
Abstract 1638: Regulation of spindle pole integrity by the MASTL-ENSA-aurora a pathway during mitosis
Authors: Kim Seul , Jun Kyoungho , Kim Ye-Hyun , Kim Jae Sung
Published: March 2024

The preservation of spindle pole integrity is crucial for proper spindle assembly and chromosome segregation in mitosis, yet the precise mechanisms governing spindle pole integrity remain elusive. During mitosis, phosphorylated Ensa (p-ENSA) localizes to spindle poles, and the inhibition of ENSA leads to a range of mitotic defects, including misaligned chromosomes, multipolar spindles, asymmetric bipolar spindles, and centrosome aberrations, resulting in a delayed mitotic progression. Remarkably, the mitotic delay induced by ENSA inhibition is alleviated upon depletion of PP2A-B55α, but spindle pole defects persist. Significantly, we have observed an interaction between ENSA and Aurora A during mitosis, and the inhibition of ENSA diminishes Aurora A expression at the mitotic spindle poles. Intriguingly, the injection of MKI-2-sensitized tumors is associated with heightened chromosomal instability and downregulation of the MASTL-ENSA-Aurora A pathway in an orthotopic breast cancer mouse model. These findings offer new insights into the regulation of spindle pole integrity through the MASTL-ENSA-Aurora A pathway during mitosis, emphasizing the pivotal role of ENSA in recruiting Aurora A to the spindle pole, independently of PP2A-B55α.

Citation Format: Seul Kim, Kyoungho Jun, Ye-Hyun Kim, Jae Sung Kim. Regulation of spindle pole integrity by the MASTL-ENSA-aurora a pathway during mitosis [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 1638.
Abstract 1665: Investigating Daple-FLT3 gene fusion in leukemia cells and its response to kinase inhibitors
Authors: Nguyen Elena , Ear Jason
Published: March 2024

Gene fusions are frequently found in leukemia and other cancers. The signaling scaffold protein, Daple (CCDC88C), has been found to undergo gene fusion to the receptor tyrosine kinase, FLT3. Specifically, a fragment of the coiled-coil domain from Daple and tyrosine kinase domain (TKD) of FLT3 are found fused together. How the gene product from this gene fusion functions in rewiring the signaling network in cells and whether this gene product can be targeted though pharmacological inhibitions remain unclear. Cell surface receptors, such as receptor tyrosine kinases (RTKs), activate cell signaling and genetic mutations that lead to constitutive receptor activation can promote unregulated cell proliferation, survival, and migration. Leukemia patients with hyperactive RTKs are treated with tyrosine kinase inhibitors (TKIs), such as Imatinib and Sorafenib. Here we demonstrate how TKIs can directly block the activation of Daple-FLT3 and prevent hyperactivation of its downstream pathways. Our studies unveil a strategy to identify other compounds which may serve as an additive, or alternative, approach to treating patients with Daple gene fusion.

Citation Format: Elena Nguyen, Jason Ear. Investigating Daple-FLT3 gene fusion in leukemia cells and its response to kinase inhibitors [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 1665.
Abstract 1668: Association of stem cell-like phenotype with isoform specific functions of AKTs in hepatocytes
Authors: Slarve Ielyzaveta , Wang Yushan , Alba Mario , He Lina , Stiles Bangyan
Published: March 2024

Introduction: Liver cancer is the sixth most common cancer and the third leading cause of cancer-related deaths worldwide. It is associated with abnormal activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which is implicated in regulation of cancer stem cells (CSCs). The present study investigated the effects of AKT loss on stem cell-like phenotype in immortalized mouse hepatocytes.

Methods: Immortalized hepatocytes were established from mice with Akt1 (Akt1−/−; Akt1-null) or Akt2 (Akt2−/−; Akt2-null) whole-body deletion. Cell morphology was assessed via microscopy. Cell proliferation was evaluated with hemacytometer count and MTT assay. Cell migration towards a chemoattractant was assessed via transwell assay. Cell viability was evaluated in response to chemotherapy treatment. The ability of cells to

Results: When compared to WT cells, which exhibited cuboidal epithelial-like features, Akt1-null and Akt2-null cells demonstrated elongated mesenchymal-like morphology. Loss of either isoform was associated with a significantly decreased cell growth (p<0.05). Akt1-null cells migrated faster towards a chemoattractant in the transwell assay, were significantly more resistant to sorafenib, and formed a significantly higher number of spheres in the sphere formation assay (p<0.01). Proteomic analysis revealed that both Akt1-null and Akt2-null cells exhibited increased expression of CSC marker CD44, along with other proteins associated with liver cancer, including PDGFRA, CDH2, MMP14, and COX2 (p<0.05). Additionally, Akt1-null cells exhibited elevated levels of ANXA1 and ANXA3 (p<0.01).

Conclusions: Loss of both AKT isoforms resulted in decreased cell proliferation and elevated expression of proteins with a previously reported role in liver cancer invasion, migration, and epithelial-mesenchymal transition. AKT1 loss correlated with an enhanced stem cell-like phenotype, including increased migration towards a chemoattractant, higher resistance to chemotherapy, and enhanced ability to form spheres. These results contribute to the understanding of the role of AKT isoforms in regulating stemness in liver cancer and may serve as a basis for investigation into isoform specific inhibitors. Further investigation into cellular mechanisms governing these phenotypes is required.

Citation Format: Ielyzaveta Slarve, Yushan Wang, Mario Alba, Lina He, Bangyan Stiles. Association of stem cell-like phenotype with isoform specific functions of AKTs in hepatocytes [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 1668.
Abstract 1685: Investigating PAX8 interactions in ovarian cancer
Authors: Sereti Kostianna , Russo Anna , Raisner Ryan , Gascoigne Karen
Published: March 2024

Ovarian cancer is the most lethal gynecological cancer and there is a critical unmet need to develop novel therapies. The transcription factor, PAX8, is upregulated in ~80% of renal, ovarian, endometrial and thyroid tumors and its overexpression correlates with higher risk of death and recurrence. PAX8 represents a promising target because of its high specificity and low expected toxicity, but how it promotes tumorigenesis remains elusive. Using proximity labeling followed by mass spectrometry, we identified a list of PAX8 binding partners and have further confirmed these interactions at the genomic level through ChIP-seq and ATAC-seq. Our findings reveal novel PAX8 interactions and a potential therapeutic approach for ovarian cancer treatment.

Citation Format: Kostianna Sereti, Anna Russo, Ryan Raisner, Karen Gascoigne. Investigating PAX8 interactions in ovarian 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 1685.
Abstract 1739: Detection of regulatory T cells, a comparison of flow cytometry and epigenetic qPCR immunophenotyping using healthy donor and oncology patient whole blood
Authors: Baron Udo , Lozza Laura , Bisconte Angelina , Raschke Eva , Phippard Deborah , Olek Sven
Published: March 2024

Human regulatory T cells (Tregs) are part of the adaptive immune response and play a pivotal role in suppressing both the immune and anti-tumor responses. Infiltration of Tregs into the tumor microenvironment has been associated with poor clinical prognosis. In addition, increased peripheral Treg frequency is associated with disease progression. Monitoring Tregs in both the blood and in the tumor of patients is being used to evaluate clinical prognosis and response to treatment in immuno-oncology studies. The key challenge is the ability to detect Tregs with high accuracy and precision. Here, we have used epigenetic qPCR (Epiontis ID®) as a method to detect and quantify Tregs in peripheral whole blood and compared it with flow cytometry. Flow cytometry is the current gold standard for immunophenotyping. For method comparison, we analyzed 113 whole blood samples from healthy donors. We have demonstrated equivalence and shown a strong correlation between epigenetic qPCR and flow cytometry-based data, indicating that Epiontis ID® is a robust and sensitive DNA-based method for Treg quantification. This epigenetic qPCR method does not require fresh samples or intact cells, since DNA can be extracted from frozen blood. We then evaluated Treg levels in blood samples from solid tumor cancer patients by epigenetic qPCR to demonstrate the clinical application and relevance for immune monitoring. Here, Treg frequencies were reported as relative (%) and absolute counts (cells/µL) values. Epigenetic qPCR for immune monitoring mitigates the operational and technical challenges of monitoring Tregs in the periphery for immuno-oncology studies which traditionally been performed by flow cytometry. This Epiontis ID® method provides the sensitivity and accuracy needed for determining patient response in progressive disease.

Citation Format: Udo Baron, Laura Lozza, Angelina Bisconte, Eva Raschke, Deborah Phippard, Sven Olek. Detection of regulatory T cells, a comparison of flow cytometry and epigenetic qPCR immunophenotyping using healthy donor and oncology patient whole blood [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 1739.
Abstract 1753: SHOC2 is a genetic dependency in NRAS-mutant melanoma
Authors: Gu Andrea Y. , Lee Tet-Woo , Khan Aziza , Hunter Francis W. , Singleton Dean C. , Jamieson Stephen M. F.
Published: March 2024

Melanoma is the most lethal form of skin cancer, with over 300,000 new cases worldwide every year. Specifically, melanoma with NRAS mutations are of particular clinical concern due to their association with a poor prognosis and lack of specific treatment options. Therefore, there is a pressing need for novel approaches to address the treatment of NRAS-mutant melanoma. A current approach to identify novel drug targets is based on the genetic concept of induced essentiality, where functional interactions that occur in response to oncogene addiction create a dependency on another gene. To identify genetic dependencies in NRAS-mutant melanoma, whole-genome CRISPR-Cas9 knockout screens were conducted in 6 NRAS-mutant and 7 NRAS-wild-type (wt) early-passage New Zealand melanoma (NZM) cell lines that were established and maintained at 5% oxygen to mimic physiological oxygen conditions. The NZM cell lines were stably transduced with the whole-genome Brunello lentiviral single guide (sg) RNA library and screened for up to 35 days. BAGEL (Bayesian Analysis of Gene Essentiality) analyses of the NZM whole-genome knockout screens, alongside CRISPR-Cas9 screening data using the Avana sgRNA library from an additional 28 melanoma cell lines, available on the Cancer Cell Line Encyclopaedia (CCLE) database, revealed prospective gene candidates that exhibit greater detrimental effects on the fitness of NRAS-mutant cell lines compared to the NRAS-wt lines. These genes are being further validated as essential genes for NRAS-mutant melanoma cells through custom sgRNA library knockout screens and in vitro individual gene knockout studies. In particular, we demonstrate that knockout of SHOC2, a gene that encodes a scaffold protein essential for activation of the MAPK signalling pathway, results in the prevention of ERK phosphorylation and a more substantial reduction in cell growth in NRAS-mutant NZM cell lines when compared to NRAS-wt lines. These results support previous studies that have identified SHOC2 as a potential therapeutic target for RAS-driven cancers and suggest that targeting of SHOC2 may have utility in NRAS-mutant melanoma, where greater treatment options are urgently needed.

Citation Format: Andrea Y. Gu, Tet-Woo Lee, Aziza Khan, Francis W. Hunter, Dean C. Singleton, Stephen M. F. Jamieson. SHOC2 is a genetic dependency in NRAS-mutant 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 1753.
Abstract 1798: Glyoxalase 1 (GLO1) as an oncometabolic enabler of prostate cancer progression: CRISPR/Cas9-based GLO1-deletion from human DU145-Luc2 cells blocks EMT and bone metastasis in SCID mice
Authors: Jandova Jana , Cress Anne E. , Wondrak Georg T.
Published: March 2024

Glyoxalase 1 (encoded by GLO1) is a glutathione-dependent enzyme detoxifying the glycolytic byproduct methylglyoxal (MG), an oncometabolite involved in metabolic reprogramming. Recently, we have demonstrated that GLO1 is overexpressed in human prostate cancer cells and patient tumors. In order to inform the ongoing debate on the role of GLO1 as an oncometabolic enabler of tumor glucose metabolism and malignant progression, we performed CRISPR/Cas9-based GLO1-deletion in human DU145-Luc2 malignant prostate cancer cells. NanoString nCounterTM ('PanCancer-Progression-Panel') comparative gene expression profiling (GLO1_KO versus GLO1_wt) revealed a significant downregulation of EMT-related pathways in GLO1_KO cells; concordantly, phenotypical screening indicated a pronounced attenuation of matrigel invasiveness observable in GLO1_KO cells. Likewise, inclusion of MG or a small molecule GLO1 inhibitor (TLSC-702) blocked invasiveness of DU145 GLO1_wt cells. Downregulation of EMT-related genes (including MMP3, SPP1, CXCL8) was accompanied by increased expression of TXNIP (thioredoxin-interacting protein), a master regulator of cellular energy metabolism and redox homeostasis. In a bioluminescent SCID mouse bone metastasis model (intracardial injection of DU145-Luc2 GLO1_wt and GLO1_KO cells), GLO1 expression was necessary to cause mandibular bone metastases (as evidenced by the complete absence of bone metastases after GLO1 deletion). Given the availability of drug-like small molecule inhibitors of GLO1 enzymatic activity these data suggest that GLO1 represents a novel molecular target for the pharmacological suppression of prostate cancer bone metastasis.

Citation Format: Jana Jandova, Anne E. Cress, Georg T. Wondrak. Glyoxalase 1 (GLO1) as an oncometabolic enabler of prostate cancer progression: CRISPR/Cas9-based GLO1-deletion from human DU145-Luc2 cells blocks EMT and bone metastasis in SCID mice [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 1798.
Abstract 1945: Mechanisms of acquired resistance to EGFR tyrosine kinase inhibitor osimertinib and KRAS-G12C inhibitor sotorasib in lung cancer
Authors: Yagami Yuri , Sato Takashi , Yamamoto Hiroki , Shirasawa Masayuki , Nakahara Yoshiro , Hideo Watanabe , Katsuhiko Naoki
Published: March 2024

A series of molecular targeted drugs have been developed for the treatment of non-small cell lung cancer (NSCLC) harboring driver gene alterations. Although the use of these drugs has substantially improved the clinical outcomes of patients with the genetically altered NSCLC, these patients ultimately develop resistance to the drugs. Various molecular mechanisms of resistance to the molecular target therapies have been reported, it is still a crucial challenge to overcome the drug resistance. Osimertinib, the most used third-generation EGFR tyrosine kinase inhibitor to target both common EGFR mutations and secondary T790M mutation, and sotorasib, a recently developed KRAS inhibitor to target KRAS G12C mutation, are no exception. In this study, we aimed to investigate the mechanisms of acquired resistance to osimertinib and sotorasib in NSCLC by profiling drug-resistant lung cancer cell lines, which were established from EGFR-mutant HCC827(del E756_A750) and KRAS G12C-mutant H23 through exposure to each of the molecular targeted drugs. Phospho-RTK array and western blot analyses revealed MET overexpression and phosphorylation in the osimertinib-resistant HCC827 (HCC827OR) as previous studies reported. Notably in our study, HCC827OR cells were incompletely sensitive to a selective MET inhibitor savolitinib without osimertinib, suggesting that HCC827OR partially lost its dependency on mutant EGFR and got addiction to MET signaling. When sotolasib was administered to the sotolasib-resistant H23 (H23SR) cells, KRAS-GTP, and the downstream ERK and AKT phosphorylation were suppressed. To identify novel mechanisms of the resistance, we performed epigenomic profiling of active enhancer in those cells and found a candidate transcription factor, at whose gene locus H3K27 acetylation signal was increased in H23SR cells compared to parent H23 cells. Immunoblotting showed that protein expression of the gene was indeed higher in H23SR cells than the parent cells. To confirm the involvement of this gene to the resistance, further investigation is warranted.

Citation Format: Yuri Yagami, Takashi Sato, Hiroki Yamamoto, Masayuki Shirasawa, Yoshiro Nakahara, Watanabe Hideo, Naoki Katsuhiko. Mechanisms of acquired resistance to EGFR tyrosine kinase inhibitor osimertinib and KRAS-G12C inhibitor sotorasib in 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 1945.
Abstract 1953: Posttranslational modification in glucocorticoid receptor in treatment resistant prostate cancers
Authors: Gulla Surendra , Gardner Ephraim , Sundar Shreya Shyam , Jaiswal Praveen , Gao Ning , Bennet Lynda , Conzen Suzanne , Adelaiye-Ogala Remi
Published: March 2024

Prostate Cancer (PCa) remains the most common cancer in men in the United States and the second leading cause of cancer-related death in men. An evolving concept that contributes to our understanding of resistance to androgen deprivation therapy (ADT) and AR antagonists in advanced PCa is the ability of cells to turn on compensatory hormone receptor signaling for survival. Preclinical and clinical studies demonstrate that induction of glucocorticoid receptor (GR) expression confers resistance to AR-targeted therapy. Previous studies from our lab demonstrated that the inhibition of AKT signaling blocks pro-oncogenic GR activity and overcomes resistance to AR-targeted therapy. AKT1 is predicted to phosphorylate GR at s134, however, the implications of pGR(s134) in advanced PCa remains unclear. Although ligands (e.g., Dex) activates GR, we have found GR activation modulated by post-translational modification (PTM) and persistent in treatment resistant PCa. Therefore, we hypothesize pro-oncogenic GR activation through AKT1-induced PTM. The rationale is that by inhibiting AKT signaling, we block residual GR signaling that can potentially drive lethal prostate cancers and consequently be re-sensitized to the standard of care. We validated our findings by Western blot analysis, we used an AKT1-specific inhibitor (AKT1i) and observed decreased AKT1 activity and decreased pGR(s134) without affecting total GR protein expression. Functional studies using shRNA against AKT1 showed reduced pGR(s134) expression without affecting the total GR expression Our preliminary data suggests that pro-oncogenic GR activity is modulated through the phosphorylation of GR on s134 by AKT1.

Citation Format: Surendra Gulla, Ephraim Gardner, Shreya Shyam Sundar, Praveen Jaiswal, Ning Gao, Lynda Bennet, Suzanne Conzen, Remi Adelaiye-Ogala. Posttranslational modification in glucocorticoid receptor in treatment resistant prostate cancers [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 1953.
Abstract 1967: IN-119873, the next-generation allosteric EGFR TKI, a potent and highly selective EGFR L858R for the treatment of osimertinib-resistant NSCLC
Authors: Choi Jong Ryoul , Yoon Daseul , Kim Seo Hyun , Kim Do Gyeong , Kang Seock Yong , Choi Seong-Il , Park Somyi , Lee Hyun Kyung , Kim Donghyun , Lee Ju Hyun , Kim Hye-Jung , Kim Bong Tae
Published: March 2024

EGFR mutations occur in approximately 50% of Asian patients and 20% of Caucasian patients with non-small cell lung cancer (NSCLC). The EGFR L858R mutation constitutes approximately half of the EGFR-mutated patients, and despite receiving first-line or second-line treatment with the 3rd generation EGFR TKI osimertinib, they continue to exhibit a poor prognosis. According to real-world analysis, the leading resistance mechanism to osimertinib is identified as EGFR C797S mutation. Furthermore, osimertinib is still associated with gastrointestinal and skin toxicity, despite its improved selectivity for EGFR WT compared to 1st-generation EGFR TKIs. Here, we show that IN-119873 can overcome C797S-mediated acquired resistance based on the results obtained by various in vitro and in vivo NSCLC models. IN-119873 binds to an EGFR allosteric site separate from the ATP-binding site with a remarkable preference for highly mutant-selective inhibition. It effectively targets EGFR L858R activating mutations while sparing the inhibition of EGFR WT, which sets it apart from ATP competitive inhibitors. Moreover, oral administration of IN-119873 showed excellent anti-tumor efficacy in a dose-dependent manner in a diverse of osimertinib-resistant CDX and PDX models. IN-119873 also demonstrated favorable blood-brain-barrier penetration with promising intracranial efficacy in the H1975-luc brain metastasis model both mono or in combination with osimertinib. As a selective, orally available, and CNS-penetrable next-generation allosteric EGFR-TKI, IN-119873 is currently undergoing IND-enabling studies and has the potential to demonstrate activity in both first-line and resistance settings, either as a monotherapy or in combination with 3rd generation EGFR TKI.

Citation Format: Jong Ryoul Choi, Daseul Yoon, Seo Hyun Kim, Do Gyeong Kim, Seock Yong Kang, Seong-Il Choi, Somyi Park, Hyun Kyung Lee, Donghyun Kim, Ju Hyun Lee, Hye-Jung Kim, Bong Tae Kim. IN-119873, the next-generation allosteric EGFR TKI, a potent and highly selective EGFR L858R for the treatment of osimertinib-resistant NSCLC [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 1967.