bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2814: Rectal implantation paves the path to evaluate the metabolic machinery responsible for CRC metastasis
Authors: Afify Said M. , Shen Chen , Merugu Siva B. , Wynn Daniel T. , Nayak Anmada , Lu Wenhui , Calderin Ricardo M. , Pannkuk Evan , Hong Sung H. , Ayad Nagi G. , Robbins David J.
Published: March 2024

Introduction: Colorectal carcinoma patients die most often from distant metastases in liver and lung. It is crucial to develop metastatic models for the CRC in order to better understand the process, which will help in treatment.

Method: Using orthotropic transplantation techniques, we tried to test whether rectal implantation could produce spontaneous metastases in-vivo mimicking colorectal cancer. The rectum was injected orthotopically with 0.2 × 106 HCT116 cells. In order to confirm metastasis, histopathological examinations were performed. Further, we generated cancer organoids to evaluate population susceptibility to CRC metastasis and metabolic mechanisms responsible for metastasis process.

Results: We found that rectal implantation was an effective method for simulating CRC cancer metastasis using H&E staining and immunohistochemistry. In less than four weeks after the implant, HCT116 spontaneously metastasized to the liver, lungs, and D lymph node. When compared with cancer organoids derived from primary tumors, liver and lung metastases organoids proliferated more often, were more aggressive, and displayed distinct characteristics. Using non-targeting metabolomic analysis, it was found that metastases derived organoids and primary tumors derived organoids were distinguished by novel metabolites.

Conclusion: By implantation in the rectal cavity, we can evaluate the metabolic machinery that leads to CRC metastasis. In the future, implantation of patient-derived samples will become increasingly important.

Citation Format: Said M. Afify, Chen Shen, Siva B. Merugu, Daniel T. Wynn, Anmada Nayak, Wenhui Lu, Ricardo M. Calderin, Evan Pannkuk, Sung H. Hong, Nagi G. Ayad, David J. Robbins. Rectal implantation paves the path to evaluate the metabolic machinery responsible for CRC 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 2814.
Abstract 2829: Strategies to overcome challenges in randomization and grouping methodologies in in vivo studies using the power of animal study workflow software
Authors: Ramachandra Raghu
Published: March 2024

Preclinical research in Oncology requires using a wide variety of animal models. Robust, clinically relevant distribution methods including randomized and non-random methods appropriate to the model are essential to ensure unbiased group assignments and reliable experimental outcomes. This presentation will explore flaws and inconveniences in prevalent randomization methods and strategies to overcome them using animal study workflow software and demonstrate the power of each with unique examples. Strategies for randomizing individual animals into groups and randomizing mouse cages into groups will be explored. Strategies for exclusion criteria, robust documentation, multi-parameter randomization, re-randomization, and clinical relevance of these strategies will be discussed. Standardization and automation of distribution methods help improve process integrity, research quality, and reproducibility.

Citation Format: Raghu Ramachandra. Strategies to overcome challenges in randomization and grouping methodologies in in vivo studies using the power of animal study workflow software [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 2829.
Abstract 2839: The mouse models of human cancer database (MMHCdb)
Authors: Begley Dale A. , Krupke Debra M. , Jocoy Emily , Neuhauser Steven B. , Bult Carol J.
Published: March 2024

The laboratory mouse plays a pivotal role as an experimental system for investigations into the genetic and genomic basis of cancer susceptibility, initiation, and progression and for preclinical research on novel treatment strategies for cancer. Advances in mouse models include humanized mice, transplant compliant mouse strains, and panels of genetically diverse mice (e.g., Diversity Outbred and Collaborative Cross). The distributed and heterogenous nature of information about these model systems presents a significant challenge to finding and aggregating information about mouse models and to identifying the most relevant models for a particular research question. The Mouse Models of Human Cancer database (http://tumor.informatics.jax.org) is an expertly curated FAIR-compliant knowledgebase that applies community annotation standards and nomenclature standards for genes, alleles, and strains to support accurate and comprehensive search results for mouse models of human cancer. The data in MMHCdb are curated from peer-reviewed scientific publications and direct data submissions from individual investigators and large-scale programs. A primary focus of the resource is to reflect the impact that genetic background has on the cancer-related characteristics of mouse models. The database includes information on spontaneous and genetically engineered cancer models as well as Patient Derived Xenografts and diversity panels. The database supports faceted searching for models by multiple criteria including, tumor type, genetic background of the mouse model, somatic mutations, and publication. Model descriptions include summaries of observed frequencies of tumors observed in the model and histopathology images. Information in MMHCdb is linked to cancer model data from other bioinformatics resources including the Patient Derived Cancer Models database (PDCM), PathBase, the Gene Expression Omnibus (GEO), and ArrayExpress.

MMHCdb is supported by NCI R01 CA089713

Citation Format: Dale A. Begley, Debra M. Krupke, Emily Jocoy, Steven B. Neuhauser, Carol J. Bult. The mouse models of human cancer database (MMHCdb) [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 2839.
Abstract 2872: GLUT3 promotes metabolic plasticity that drives radiation resistance in glioblastoma
Authors: Abdulrahman Lobna , Bailleul Justine , Hoang Hanna , Tang Aaron , Vlashi Erina
Published: March 2024

Glioblastomas (GBM) are the most common and malignant brain tumors in adults. While radiation therapy (RT) offers a significant advantage for GBM patients, improving long-term survival remains elusive largely due to the remarkable resistant nature of these tumors. We have shown that metabolic plasticity, especially rewiring of glucose metabolism, promotes radiation survival in GBM. In this study, we investigate the role of glucose transporter 3 (GLUT3) translocation to the cell surface in radiation resistance of GBM. The effect of radiation on GLUT3 total protein levels was determined in patient-derived gliomaspheres via westerns blots, while cellular localization of GLUT3 was determined via immunofluorescence, flow cytometry and cell fractionation assays. The role of the AMPK and αvβ3 integrin in the radiation-induced GLUT3 translocation to the cell membrane was probed via small molecule inhibitors and activators. To determine the role of GLUT3 in radiation survival, GLUT3 levels were knocked down via siRNA and shRNA, followed by functional in vitro and in vivo assays. We provide evidence that irradiated gliomaspheres translocate GLUT3 to the plasma membrane, in a radiation-induced oxidative stress-dependent manner, effectively increasing their glucose uptake and promoting radiation survival. Our data also suggest that GLUT3 translocation to the cell surface contributes to RT resistance. AMPK and αvβ3 integrin are linked to radiation-induced translocation of GLUT3 to the cell membrane, suggesting that targeting these pathways is a potential approach to sensitize GBM tumors to radiation therapy.

Citation Format: Lobna Abdulrahman, Justine Bailleul, Hanna Hoang, Aaron Tang, Erina Vlashi. GLUT3 promotes metabolic plasticity that drives radiation resistance in glioblastoma [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 2872.
Abstract 2882: Investigating mechanisms of radioresistance in TYK2 knockout head and neck cancer cells
Authors: Constante Nicole Mayerli , Kelley Vanessa , Contessa Joseph
Published: March 2024

Radiation therapy is the primary form of treatment of head and neck squamous cell carcinoma (HNSCC). However, more than half of patients experience disease progression with standard of care treatment. To investigate novel therapeutic targets, a CRISPR screen was done to identify genes that are associated with radioresistance. The screening results indicated that JAK1 and TYK2 knockout (KO) resulted in radioresistance in HNSCC. Preliminary data suggests that loss of JAK1 causes an enhanced G2/M arrest phenotype and enhanced genome stability that contribute to radioresistance. To investigate whether TYK2 KO caused changes to the cell cycle and genome stability as observed in JAK1 KO, Western blots, cell cycle assays, and micronuclei staining experiments were performed. Cal27, a HNSCC cell line, was used. Cal27 cells were transduced with a non-targeting control (NT2) guide RNA (gRNA) or two different gRNAs targeting TYK2 to generate TYK2 KO cells. Western Blot analysis was performed to determine changes in signaling upon TYK2 KO. Additional conditions included radiation and treatments with Interferon gamma, Interferon alpha and Interleukin-6 to activate the canonical JAK-STAT pathways. Overall, there were minimal changes in downstream signaling between the treatments and the knockouts. Cell cycle analysis via flow cytometry revealed no changes between the NT2 and TYK2 KO cells, potentially corroborating signaling results. Forty percent of TYK2 KO cells were arrested at the G2/M checkpoint at 8 hrs and 24 hrs after radiation treatment, but twenty percent returned to the G1 phase at 24 hrs. This indicates that loss of TYK2 is not responsible for cell cycle arrest. Lastly, micronuclei formation was observed to measure genomic instability. Twenty thousand Cal27 NT2 and TYK2 KO cells were seeded. They were allowed to incubate for 48 hrs before treatment with 4 Gy. Micronuclei staining revealed minimal differences between NT2 and TYK2 KO cell lines, although there were significant differences between the control and the radiation groups. Overall, radioresistance by TYK2 KO is not caused by changes in cell cycle or genomic instability as demonstrated with JAK1 KO. To understand how TYK2 is causing radioresistance in HNSCC, next steps would be to see if there are changes in proliferation. If MTT assays demonstrate significant changes, downstream signaling in growth pathways will be measured. If there is no increased proliferation, we will examine changes in DNA damage repair.

Citation Format: Nicole Mayerli Constante, Vanessa Kelley, Joseph Contessa. Investigating mechanisms of radioresistance in TYK2 knockout head and neck cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2882.
Abstract 289: Temporal and spatial dynamics of glial cells apolipoprotein E expression in astrocytoma microenvironment
Authors: Chien Ting-Yi , Chiang Chi-Shiun
Published: March 2024

Introduction: Brain tumors have proven challenging to treat due to the complex and sensitive architecture of the brain. Previous research has shown that GBM cells depend highly on cholesterol uptake, and apolipoprotein E (APOE) is required to transport cholesterol among tissues or cells. APOE is primarily synthesized by astrocytes and has been extensively studied in neurodegenerative diseases. While extensively studied in neurodegenerative diseases, limited studies are focusing on their roles in brain tumor development. Here, we analyzed the temporal and spatial expression of APOE during brain tumor progression in a murine astrocytoma tumor model.

Material and method: ALTS1C1 astrocytoma cells were intracranially injected into the mouse brain. The animal was sacrificed, and the left hemisphere, right hemisphere, and tumor tissue were collected for flow cytometry analysis. CD45, CD11b, and GFAP were used to recognize macrophages, microglia, and astrocytes. CD68, TMEM119, and GFAP were used for tissue immunostaining to label macrophages, microglia, and astrocytes, respectively.

Results and Discussion: Flow cytometry analysis revealed upregulated APOE in tumor-infiltrating microglia, macrophages, and lymphocytes. GFAPhigh astrocytes displayed lower APOE levels than GFAPlow astrocytes. IHC analysis showed that CD68+ macrophages exhibited higher APOE expression at the tumor edge than normal tissue. On the contrary, GFAP+ astrocytes at the tumor edge displayed a lower APOE signal than the normal tissue. Previous studies have shown the participation of APOE in regulating immune response in various cancers. Further studies to analyze the dynamic change of APOE on the gene expression level with in situ hybridization and the quantification of cholesterol levels in the microenvironment may help uncover their roles in the glioma microenvironment.

Conclusion: This study demonstrated that APOE was temporal and spatial differentially expressed in macrophages and astrocytes in the brain tumor microenvironment. Tumor-infiltrating cells showed an elevated APOE expression, while GFAP+ astrocytes expressed reduced APOE levels. Although the mechanism of these alterations remains unclear, this study provides a basis for future studies about the roles of APOE in the brain tumor microenvironment.

Citation Format: Ting-Yi Chien, Chi-Shiun Chiang. Temporal and spatial dynamics of glial cells apolipoprotein E expression in astrocytoma 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 289.
Abstract 2903: CTHRC1 induces PSCs (pancreatic stellate cells) into myCAFs (myofibroblast-like cancer-associated fibroblasts)
Authors: Lee Jin Gyeong , Kang Min Kyung , Jiang Fen , Ryu Kyoungjin , Kim Juwon , Park Yun-Yong , Koh Sang Seok
Published: March 2024

Collagen triple helix repeat containing-1 (CTHRC1) is a secreted protein that contributes to the progression of various cancers, including pancreatic cancer. The higher expression of CTHRC1 in tumor tissues is associated with poorer survival outcomes. However, its specific roles in tumor extracellular matrix (ECM) remodeling remain unclear. Our study aims to investigate the influences of CTHRC1 on pancreatic stellate cells (PSCs), a main source of ECM production in pancreatic cancer. The analyses of the publicly available pancreatic cancer patient data revealed that CTHRC1 is mainly expressed in cancer stroma and highly correlated with ECM-related genes. An in vitro study showed that more than 40% of these genes can be upregulated by CTHRC1. CTHRC1 specifically activated PSC into myofibroblast-like cancer-associated fibroblasts (myCAFs), which are characterized by a significantly upregulated POSTN gene expression. Periostin (coded by the POSTN gene) has a central role in the CTHRC1-PSCs-cancer metastasis axis. Furthermore, CTHRC1 promoted pancreatic cancer cell proliferation through PSC activation to a greater extent than via direct stimulation. Proof-of-concept experiments showed that the long-term (4-week) inhibition of CTHRC1 led to significant tumor suppression and ECM reduction, and also resulted in an unexpected shift in the CAF subtype from myCAFs to inflammatory CAFs (iCAFs). PSC activation was demonstrated to be the key molecular mechanism responsible for the tumor-promoting effects of CTHRC1, and CTHRC1 has a critical role in CAF subtype differentiation and tumor microenvironment (TME) remodeling. The inhibition of CTHRC1 as a therapeutic strategy for the treatment of pancreatic cancer warrants further investigation.

Citation Format: Jin Gyeong Lee, Min Kyung Kang, Fen Jiang, Kyoungjin Ryu, Juwon Kim, Yun-Yong Park, Sang Seok Koh. CTHRC1 induces PSCs (pancreatic stellate cells) into myCAFs (myofibroblast-like cancer-associated fibroblasts) [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 2903.
Abstract 2943: Investigation of microsatellite instability in RNA compared to DNA, using microsatellite targeted, anchored multiplex PCR and NGS
Authors: Rogge Ryan , Patterson Taylor , Navetta Alicia , Legare Dhruti
Published: March 2024

MSI measurements are typically made on DNA, but it would be useful to make similar measurements from RNA or total nucleic acid inputs (TNA), so that MSI determinations could be made in parallel to interrogation of the RNA. Doing so requires characterizations of microsatellite lengths from those which are transcribed and untranscribed, because both will be detected following cDNA creation. Microsatellite instability (MSI) occurs when small unit repeats of DNA undergo incorrect replication resulting in the growth or contraction of the number of repeats in the newly synthesized DNA. This accumulation of errors at microsatellite DNA is usually mitigated by the mismatch repair (MMR) pathway which can correct these errors. MSI is of interest because it serves as a phenotypic readout of the status of the MMR machinery, and MSI status can correlate to tumor progression and response to agents that regulate these cellular mechanisms. We investigated the status of microsatellites using Anchored Multiplex PCR (AMP™) targeted panels (RUO) and compared results from TNA inputs using an AMP MSI module and both VARIANTPlex™ chemistry to interrogate DNA, and FUSIONPlex™ chemistry to interrogate both RNA + DNA. Our results indicate differences in the diversity of lengths of microsatellites present in RNA, and the resulting stability calls which would be made at those sites. Microsatellite length diversity was highly dependent on the genomic context of the microsatellite. Microsatellites located within transcripts showed increased length diversity when examining the RNA + DNA results compared to DNA alone. Microsatellites located in intergenic regions, which are unlikely to be transcribed, showed no increase in diversity of lengths when comparing the RNA + DNA results to DNA alone. While these results suggest that MSI determinations could be made from RNA or RNA + DNA NGS data, the unique behavior of microsatellites in RNA is likely to require additional investigations and unique data analysis techniques to correctly categorize microsatellites as stable or unstable.

Confidential - Company Proprietary

Citation Format: Ryan Rogge, Taylor Patterson, Alicia Navetta, Dhruti Legare. Investigation of microsatellite instability in RNA compared to DNA, using microsatellite targeted, anchored multiplex PCR and NGS [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 2943.
Abstract 2944: A high-throughput screening system for identifying therapeutic targets in immuno-oncology
Authors: Parikh Dippal , Choi Soon Youn , Pyo Ki Eun , Lee Hong-Pyo , Han Kyuho
Published: March 2024

INTRODUCTION: Over the last decade, cancer immunotherapy has revolutionized cancer treatment. However, more than half of patients don’t respond, and responders often lack lasting effects. Understanding how genetic variants in cancer cells influence their interaction with immune cells, especially T cells, is crucial to uncover underlying mechanisms. Genetic screens are powerful for uncovering responsible genes for various phenotypes, but challenges in obtaining ample samples to set up a proper immune and cancer co-culture system have limited their use for identifying immune modulators in cancer: primary mouse T cells expressing CD3 against a specific antigen (typically albumin) need to be extracted from transgenic mice and only syngeneic mouse cancer cell lines that match these T cells can be paired, restricting the utilization of human T cells and human cancer cells for this co-culture screening setup.

METHODS: Here, we present a highly scalable co-culture system that enables genome-wide CRISPR screens on a pair of human primary T cells and any allogeneic human cancer cell lines. This system utilizes the expression of a membrane-targeted protein in mammalian cells, named as ImBridge, which directly engages and activates CD3+ immune cells. This mechanism bypasses the need for matched MHC on target cancer cells.

RESULTS: We validated activation of CD3+ immune cells and selective cancer cell cytotoxicity by ImBridge. A genome-scale CRISPR screen utilizing the co-culture system unveiled known immune-oncology targets including PD-L1 and PTPN2 and revealed MEDiC_01 as a novel therapeutic target. Genetic loss of MEDiC_01 in cancer cells significantly sensitized them to cytotoxic cells, effectively eliminating cancers in the presence of human PBMCs.

CONCLUSION: We demonstrated a scalable and efficient approach for conducting high-throughput functional screens to uncover genes that modulate interactions between effector T cells and target cancer cells. This can facilitate the identification of new immuno-oncology therapeutic targets, insights into underlying mechanisms, and genetic factors influencing immune susceptibility and evasion.

Citation Format: Dippal Parikh, Soon Youn Choi, Ki Eun Pyo, Hong-Pyo Lee, Kyuho Han. A high-throughput screening system for identifying therapeutic targets in immuno-oncology [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 2944.
Abstract 3008: Loss of Rab25 accompanied by NRAS activation cooperatively mediates the tumorigenic transformation in melanoma
Authors: Kandel Samikshya , Rao Krishna
Published: March 2024

Background: Ras oncogene activation is a common and often essential event in cancer development, including melanoma. Ras mutation is present in 20-30% of melanomas and remains an undrugged target. NRAS mutation is the most frequent Ras isoform activated in melanoma, with more than 80% cases of Q61 substitutions, and is closely associated with tumor aggressiveness. Rab25 is a ubiquitously expressed small GTPase protein with a context-dependent role in cancer pathophysiology. Its role remains undiscovered in the case of melanoma. Rab25 deep deletion can be seen in portions of melanoma in databases such as the Cancer Genome Atlas and Cbioportal. Interestingly, Rab25 has a consensus sequence homologous with the binding site of Q61 mutant Ras. We propose that the loss of Rab25 in melanocytes accompanied by Ras oncogenic activation are significant events in melanoma development, and restoration of Rab25 can potentially inhibit constitutively activated N-RAS; Rab25 serves as a tumor suppressor in melanoma genesis.

Objectives: To discover the role of Rab25 in melanoma, we studied Rab25 expression via mRNA and protein levels in different melanoma cell lines and screened biopsy-proven melanoma samples through immunohistochemistry in 61 different melanoma sections. Further, to see the tumor suppressive mechanism of Rab25 in vitro, we transduced selected cell lines Rab25.

Results: There is an 87% and 62.7% reduction in Rab25 mRNA level in NRASQ61R(SKMEL-2) and NRASQ61L type (WM1366) melanoma cell lines, respectively, also reflected in Rab25 protein level, and statistically significant at p-value <0.005, using student’s T-test. Further, Rab25, prominent in adjacent normal tissues, was significantly lost in biopsy-proven melanoma tissues, with an H-score ranging from 13.28 to 91.77% and 0.00 to-11.24 % for normal skin tissue and melanoma section, respectively, and a negative correlation of -0.350, indicating a promising association of Rab25 loss with the progression of melanoma.

Conclusion: There is a loss of Rab25 in NRAS mutant melanoma with cancer progression. We saw a significant increase in Rab25 expression among the transduced sublines of NRAS mutant melanoma cell lines at the mRNA level, which will be further assessed through western blotting, cell proliferation assay, trans well migration assay, soft-agar colony formation assay to confirm NRAS mutant melanoma cell behavior. We will attempt to deliver a liposomally encapsulated six-peptide sequence to therapeutically inhibit the mutant NRAS.

Citation Format: Samikshya Kandel, Krishna Rao. Loss of Rab25 accompanied by NRAS activation cooperatively mediates the tumorigenic transformation in 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 3008.
Abstract 3066: Lung cancer cells overcome nutrient stress by upregulating an integrin αvβ3/AMPK/OXPHOS axis
Authors: Nam Arin , Jain Shashi , Campos Alejandro , Shepard Ryan M. , von Schalscha Tami , Weis Sara M. , Wettersten Hiromi I. , Cheresh David A.
Published: March 2024

Tumor cells are exposed to various forms of stress within the tumor microenvironment, and those cells that adapt often gain stem-like properties that facilitate tumor initiation and metastasis. Here, we show that lung cancer cells exposed to nutrient stress adapt and survive by converting their metabolism from glycolytic to oxidative phosphorylation (OXPHOS). This is based on the cell’s ability to upregulate expression of integrin αvβ3, a driver of tumor initiation, progression, and drug resistance. Mechanistically, integrin αvβ3 recruits and activates c-Src, promoting sustained AMPK activity leading to activation of PGC-1α, which promotes the expression of mitochondrial OXPHOS complexes. In fact, ectopic expression of integrin β3 was sufficient to drive OXPHOS-dependent metabolism even in the absence of stress. Furthermore, pharmacological targeting of this pathway not only rendered integrin αvβ3+ cells sensitive to nutrient depletion but also prevented their ability to undergo tumor initiation. These findings reveal that targeting integrin αvβ3 or its pathway can prevent cancer cell metabolic flexibility and their capacity to adapt to stress within the tumor microenvironment.

Citation Format: Arin Nam, Shashi Jain, Alejandro Campos, Ryan M. Shepard, Tami von Schalscha, Sara M. Weis, Hiromi I. Wettersten, David A. Cheresh. Lung cancer cells overcome nutrient stress by upregulating an integrin αvβ3/AMPK/OXPHOS axis [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 3066.
Abstract 3094: Biophysics in kinase drug discovery programs: Spectral shift technology from fragment screening to mechanism of action
Authors: Legros Celine , Porkolab Vanessa , Sigoillot Maud , Mirguet Olivier
Published: March 2024

Using Eurofins Discovery’s state-of-the art Hit-Finding platform, our experts designed an optimized screening cascade combining cutting-edge High-throughput Screening (HTS) technologies with the latest Biophysics techniques. With advanced technologies, such as the Echo® MS and RapidFire MS for HTS, and SPR, MST, TSA, nanoDSF and ITC for Biophysics, the cascade is effective for primary screening, orthogonal assays and evaluating Mechanism of Action (MoA). There are many biophysical approaches which bring value to drug discovery programs, and it is the target properties which drive the selection of the most suitable technology. In this presentation, we will highlight the novel Spectral Shift technology, one of the techniques employed by the Dianthus (well-known for the MST-TRIC). The goal was to rapidly implement an assay that monitors the ability of PIM3 to bind to ATP, as well as to compounds and fragments in solution. Using the Echo MS’ acoustic-droplet ejection, nanoliter dispensing in 394-well plates, robust data by Spectral Shift were quickly generated for the fragment-based screening campaign. Hits were confirmed in Dose-response with µM-range affinity values (KD), and TSA was used as an orthogonal assay for the identification of stabilizers among the selected PIM3 binders. For a defined set of 826 proprietary fragments, a single dose screening at 600µM using Spectral Shift was run, leading to the identification of 11% of potential binders. The 100 fragments were then confirmed in dose-response titration and 80 binders were confirmed. The top 30 binders were selected for orthogonal characterization using TSA. This led to the selection of the top 6 confirmed fragments, on which a MoA study in MST-TRIC was performed. Following this evaluation, 3 allosteric binders were identified, with KD from 355 to 550µM (affinity of orthosteric binders from 55 to 333µM). Biophysics plays an important role in drug discovery by qualifying valuable assets and increasing the success rates of future Hit-finding programs. The fragment hits selected in this PIM3 Kinase project, which show ADP-Glo™ activities, are now ready for crystallography studies (NMR & X-ray co-crystallization).

Citation Format: Celine Legros, Vanessa Porkolab, Maud Sigoillot, Olivier Mirguet. Biophysics in kinase drug discovery programs: Spectral shift technology from fragment screening to mechanism of action [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 3094.
Abstract 3097: CETSA HT: Employing rapid high-throughput target engagement technology in primary screening of P53
Authors: Kacal Merve , Friman Tomas , Arnold Laurence , Brehmer Victoria , Molina Daniel Martinez , Lundgren Stina
Published: March 2024

The confirmation of target engagement for candidate drugs is a fundamental requisite in drug discovery. A significant portion of projects fail to reach the clinic due to lack of efficacy or failure to show lead candidates interacting with the intended target in a more complex environment. The utility of using the CETSA® (CEllular Thermal Shift Assay) technology to measure target engagement in intact cells is increasingly exploited in high throughput screening, where examples demonstrate low false positive rates in screens of up to 500K compounds. The p53 tumor suppressor protein, a crucial guardian against cancer, functions as a transcription factor that mediates cancer suppression by regulating genes involved in a wide range of cellular processes such as DNA repair, cell cycle and apoptosis. Unfortunately, frequent mutations in the TP53 gene hinder p53's tumor suppressor role in human cancers, thus designating it as a promising target for anticancer therapy. However, attempts to restore p53 function for therapeutic purposes have not yet advanced to clinical approval, highlighting the complexities in leveraging p53 for cancer treatment. Its undruggable nature, attributed to the absence of accessible deep pockets, lack of enzymatic activity, and its intracellular location, further complicates targeted interventions with both low and high molecular weight drugs. Here, we demonstrate the analysis of a high-throughput plate based CETSA® HT screen on the challenging target p53, performed using AlphaLISA® SureFire® Ultra™ technology and our newly developed high-throughput screening platform. Hits identified from this screen are privileged from the onset by binding to the target in a physiologically relevant cellular matrix and in a binding-mode agnostic way. This is in contrast to traditional screening methods targeting activity or in vitro interaction where cellular activity is addressed in later stages. This assay cascade highlights a rapid target-to-hit lead generation strategy placing the emphasis on finding chemical matter that interacts with endogenous target in living cells first, followed by analysis of hit material activity. The ‘shortcut’ to chemical matter active in living cells represents significant potential cost savings in comparison to assay development time on recombinantly produced proteins and/or modified cell lines for traditional HTS screening approaches that still rely on target engagement assessment downstream.

Citation Format: Merve Kacal, Tomas Friman, Laurence Arnold, Victoria Brehmer, Daniel Martinez Molina, Stina Lundgren. CETSA HT: Employing rapid high-throughput target engagement technology in primary screening 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 3097.
Abstract 3114: Development of HTS enzymatic assays for RNA helicases: DDX3, DDX5, DDX17, RIG-1 and MDA5
Authors: Ali Mahbbat , Pham Ha , Martinez Anibal Ramos , Lowery Robert
Published: March 2024

RNA helicases are a broad class of enzymes that bind and modify RNA in an ATP-dependent manner and play diverse roles in RNA metabolism, regulation of gene expression, and innate immunity. Several RNA helicases, especially in the dead box helicase (DDX) family, have been shown to be dysregulated in cancer, and there have been limited efforts to develop small molecule inhibitors. To accelerate development of selective helicase inhibitors while avoiding off target effects, especially with helicases that promote tumor immunity, we are assembling a panel of high throughput biochemical assays using the Transcreener ADP2 assay for homogenous detection of RNA-dependent ATPase activity with a far-red fluorescence polarization (FP) readout. Here we describe development of assays for DDX3, DDX17 and DDX5, which have all been implicated in cancer, and the Rig-1-like receptor (RLR) helicases, RIG-1 and MDA5, which trigger expression of type I interferons important for tumor immunity. We produced the recombinant enzymes using BaV-infected insect cells, as full-length polypeptides, or in some cases truncated to remove disordered N-terminal domains. We determined RNA substrate and cofactor requirements and kinetic parameters with the purified enzymes and optimized the assays for initial velocity detection of RNA-dependent ATPase activity with a Z’ value greater than 0.7, ensuring a sufficient signal window for inhibitor screening and dose response assays. We validated each of the helicase ATPase assays for HTS by screening a collection of bioactives (Tocris 2.0) and confirming hits in dose response mode with the enzymatic assay and with thermal shift assays to confirm target binding. These assays will enable screening and hit-to-lead/SAR for DDX helicases that drive tumorigenesis as well as selectivity profiling to avoid off target effects with RLR helicases that promote tumor immunity.

Citation Format: Mahbbat Ali, Ha Pham, Anibal Ramos Martinez, Robert Lowery. Development of HTS enzymatic assays for RNA helicases: DDX3, DDX5, DDX17, RIG-1 and MDA5 [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 3114.
Abstract 3132: A novel dual drug antibody-drug conjugate targeting hTrop2 has synergetic anti-tumor activity in solid tumor models
Authors: Zhu Jie , Zhao Xiaobei , Li Eileen , Xu Hong , Nie Lei , Chen Gang , Wang Haibing
Published: March 2024

Antibody-drug conjugates (ADCs) are emerging therapeutic agents for targeted cancer treatment with remarkable clinical success. However, tumor heterogeneity and microenvironment complexity are still the major factors contributing to drug resistance, recurrence, and metastasis, which cannot be resolved with a mono therapy strategy. We’ve developed a novel dual drug ADC (BiADC), BR113, by conjugating an anti-hTrop2 antibody with a drug linker of a Topoisomerase I inhibitor payload, and another drug linker of an immune stimulator. Our studies show that the homogeneous ADC containing two distinct payloads is a new promising drug class, with two different anti-tumor MOAs, synergistic antigen-specific cell killing potency, desirable pharmacokinetic profiles, and minimal toxicity at therapeutic doses. In the cancer cells and human PBMC co-culture assay, BR113 exerts significantly more potent killing on cancer cells than two individual single-drug ADCs and the combination of two single-drug ADCs, which suggested a synergy on anti-tumor effects of this dual-drug ADC. In the xenograft mouse model studies, BR113 exhibits greater treatment effect and survival benefit than the combo of two single-drug ADCs at various doses. This robust superior anti-tumor activity is further confirmed in a hTrop2-MC38 syngeneic model with a prolonged anti-tumor protection against the tumor re-challenge. Finally, the novel BiADC, BR113, is well tolerated in hTop2 humanized mice after repeated administration. Our findings highlight the therapeutic potential of the BiADC format for treating refractory advanced solid tumors. The outstanding anti-tumor activity across various CDX tumor models and its favorable safety profile in mice support further evaluation of the innovative BiADC, BR113.

Citation Format: Jie Zhu, Xiaobei Zhao, Eileen Li, Hong Xu, Lei Nie, Gang Chen, Haibing Wang. A novel dual drug antibody-drug conjugate targeting hTrop2 has synergetic anti-tumor activity in solid tumor models [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 3132.
Abstract 3139: BL-M11D1, a novel CD33-targeting ADC, demonstrates robust anti-tumor efficacy in preclinical evaluation
Authors: Wan Weili , Zhao Shuwen , Zhuo Shi , Zhang Yong , Chen Lan , Li Ruigang , Khalili Jahan Salar , Sa Xiao , Yan Yongqi , Shen Xuejiao , Zhu Yi
Published: March 2024

CD33, is a transmembrane receptor expressed on cells of myeloid lineage. It is therapeutically targeted in cancer due to overexpression broadly among hematopoietic malignancies including AML. To develop a promising therapeutic anti-tumor agent, we generated BL-M11D1, a CD33-Ed-04 ADC. It is built on gemtuzumab, a specific monoclonal antibody against CD33, having a wt Fc that can mediate ADCC in vitro. BL-M11D1 is composed of gemtuzumab, a cathepsin B cleavable linker, and a novel topoisomerase I inhibitor agent (Ed-04). The novel Ed-04 is a derivative of the alkaloid camptothecin and mediates cell cycle arrest at the S phase and subsequent apoptosis. BL-M11D1 achieves a high drug-to-antibody-ratio (DAR=10) with a highly stable linker and is readily internalized and trafficked to lysosomal cellular compartments. The antitumor efficacy of BL-M11D1 was evaluated in xenograft tumor models. BL-M11D1 exhibited strong tumor inhibition capacity toward the human hematopoietic malignancies engrafted xenograft models. In summary, these studies suggest BL-M11D1, a novel CD33-targeting ADC, is potentially efficacious in the treatment of CD33-expressing disease. The clinical phase I trial is being undertaken to evaluate safety, dosing and observations of preliminary signs of efficacy.

Citation Format: Weili Wan, Shuwen Zhao, Shi Zhuo, Yong Zhang, Lan Chen, Ruigang Li, Jahan Salar Khalili, Xiao Sa, Yongqi Yan, Xuejiao Shen, Yi Zhu. BL-M11D1, a novel CD33-targeting ADC, demonstrates robust anti-tumor efficacy in preclinical evaluation [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 3139.
Abstract 3169: The role and mechanism of m6A methylation modification of LncRNA-ATB in regulating the invasion and metastasis of colorectal cancer
Authors: Lang# Yu , Lin# Jia-Lin , Kang Jia-Peng , Yang Lu , Zhou Dan , Fang* Can-Bin , Ye* Feng , Tang* Wei-wei
Published: March 2024

Background: The epigenetic modification of LncRNA N6-methyladenosine (m6A) methylation plays a wide role in the invasion and metastasis of various malignant tumors. However, the role of LncRNA-ATB m6A methylation modification in colorectal cancer (CRC) has not been systematically elucidated.

Methods: Detect the regulatory effect of ALKBH5 on LncRNA-ATB by Luciferin reporter gene. LncRNA-ATB expression was detected by IHC and qPCR in CRC tissues and cells. Transwell and scratch tests were used to detect the effect of ALKBH5 overexpression after LncRNA-ATB knockdown and replacement on the invasion and migration ability of CRC cells. Western blotting was used to detect the expression changes of MMPs proteins.

Results: ALKBH5 could regulate the methylation level of the LncRNA-ATB 3’ UTR region (p<0.05). ALKBH5 overexpression in CRC cells led to a significant decrease in the expression level of LncRNA-ATB (p<0.05). LncRNA-ATB exhibited elevated expression in CRC cells and demonstrated significantly higher levels in CRC tissues compared to paracancerous tissues (p<0.05), indicating the potential tumor-promoting effect of LncRNA-ATB in CRC, with the mechanism possibly regulated by ALKBH5-mediated m6A demethylation. ALKBH5 overexpression inhibited the proliferation and migration of CRC cells after LncRNA-ATB knockdown. However, this inhibitory effect was attenuated upon LncRNA-ATB rescue, accompanied by an increase in MMP9 and a decrease in TIMP1.

Conclusion: LncRNA-ATB plays a crucial role in ALKBH5-mediated m6A demethylation, regulating the invasion and metastasis of CRC. The ALKBH5-LncRNA-ATB m6A demethylation-MMPs signaling axis has been identified as a key regulatory pathway promoting CRC migration. m6A modification of lncRNA-ATB modulated by ALKBH5 provides a theoretical basis for novel therapeutic strategies for patients with metastatic CRC.

FUNDING: This study was supported by the National Natural Science Foundation of China (Grant No. 81702414), Natural Science Foundation of Fujian Province of China (Grant No. 2020J05306) and Xiamen Science and Technology Planning Project (Grant No. 3502Z20194003).

Citation Format: Yu Lang#, Jia-Lin Lin#, Jia-Peng Kang, Lu Yang, Dan Zhou, Can-Bin Fang*, Feng Ye*, Wei-wei Tang*. The role and mechanism of m6A methylation modification of LncRNA-ATB in regulating the invasion and metastasis of 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 3169.
Abstract 3181: Immune modulatory activity of radio-sensitizing gold nanoparticle
Authors: Kulkarni Tanmay , Dutta Shamit , Parent Hazel , Perry Karington , Mackeyev Yuri , Bhattacharya Santanu
Published: March 2024

Radiation therapy (RT) is frequently used in the neoadjuvant setting in borderline resectable and some resectable pancreatic ductal adenocarcinoma (PDAC) and in the definitive setting in locally advanced PDACs which is third leading cause of cancer associated death. Higher doses of RT improve overall treatment outcomes but increasing the dose significantly above 50Gy in standard 2Gy fractions risks profound gastrointestinal (GI) toxicity. Additionally, genotoxic stress such as RT, can induce CXCL12 secretion by cancer associated fibroblast (CAFs) and overexpression of CXC motif chemokine receptor 4 (CXCR4) in cancer cells. CXCL12-CXCR4 downstream signaling results in immune suppression and consequent enhanced tumor growth and metastasis and poor prognosis in PDAC. To overcome these clinical challenges, we have developed multimodal gold nanoparticle constructs (GNPs) by functionalizing the CXCR4 antagonists to (i) bind to CXCR4 and mediate GNP internalization, (ii) abolish the feedback immune inhibitory signals mediated by CXCR4 overexpression, and (iii) enhance therapeutic benefit of radiation. To achieve these multimodal activities, we have covalently attached the CXCR4 antagonist, AMD070, to thiol-terminated polyethylene glycol (PEG) to synthesize AMD-PEG which was then conjugated with GNPs to produce AMD-PEG-GNP. In our experiment AMD-PEG-GNP has successfully inhibited CXCL12-CXCR4 downstream signalling such as reduction of ERK phosphorylation in PDAC cells. To analyze the radiosensitization role of GNP nanoconjugates, PDAC cells were incubated with AMD-PEG, PEG-GNP and AMD-PEG-GNPs for 4 hours and then treated with increasing doses of radiated, from 1-4Gy. AMD-PEG-GNP demonstrated a systematic enhancement on reactive oxygen species (ROS) production and downregulation of a colony formation. Collectively, the results from in vitro study with PDAC cells confirm that the nanoconjugate with AMD070 enhances radio-cytotoxicity and impair CXCL12-CXCR4 downstream signaling. Hence AMD-PEG-GNP has strong merit to enhance radiation dose locally and simultaneously restrict RT induced immunosuppression in PDAC.

Citation Format: Tanmay Kulkarni, Shamit Dutta, Hazel Parent, Karington Perry, Yuri Mackeyev, Santanu Bhattacharya. Immune modulatory activity of radio-sensitizing gold nanoparticle [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 3181.
Abstract 3182: Role of Frizzled 7 in melanoma development and metastasis
Authors: Nihal Minakshi , Umar Sheikh A. , Chang An-Bo A. , Ahmad Nihal , Chang Hao
Published: March 2024

The frizzled (Fzd) family of proteins are 7-transmembrane G-protein coupled receptors (GPCRs) that transduce signals mainly for Wnt ligands. There are 10 Fzds identified in mammals, and they are involved in multiple cellular and biological functions via signaling through the canonical Wnt/beta-catenin, planar cell polarity, and calcium signaling pathways. Our previous work revealed a pro-metastasis role of Fzd6 in melanoma. This project focuses on Fzd7, a homolog of Fzd6, in melanoma. We found that FZD7 is highly expressed in multiple melanoma cell lines. siRNA knockdown of FZD7 does not affect cell proliferation but significantly reduces cell invasion in A375, Hs294T, and SK-MEL28 cells in vitro. To determine the in vivo role of Fzd7 in melanoma, we generated Fzd7 knockout YUMM1.7 cell lines for xenograft experiments using CRISPR. All three Fzd7 knockout clones showed a significant reduction in primary tumor growth when xenografted in C57BL/6 mice. To determine the potential mechanism of Fzd7 in melanoma growth and invasion, we performed bulk RNA-Seq on the YUMM1.7 knockout cell lines and identified 215 downregulated and 31 upregulated genes (with a fold change of 2, p<0.05). We are in the process of confirming these transcriptional profile changes. Further functional analysis will be performed using gain- and loss-of-function approaches to determine the downstream mechanisms of Fzd7 in melanoma growth and invasion.

Citation Format: Minakshi Nihal, Sheikh A. Umar, An-Bo A. Chang, Nihal Ahmad, Hao Chang. Role of Frizzled 7 in melanoma development and 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 3182.