bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 171: Prostate cancer organoids from KUCaP patient-derived xenograft(PDX) series enables us to genetic engineering human-derived cellular models
Authors: Sunada Takuro , Goto Takayuki , Sumiyoshi Takayuki , Shusuke Akamatsu , Kobayashi Takashi
Published: June 2023

Introduction and Objectives Cancer organoids are expected to be valuable models that recapitulate intratumor heterogeneity and the phenotypic diversity of the disease ex vivo. However, the establishment rate of prostate cancer (PCa) organoids directly from clinical specimens are still low, and we need to improve. Patient-derived xenograft model (PDX) is widely used as a human-derived in vivo model, but genetic engineering is impossible, limiting its use. We aimed to develop a method to stably grow prostate cancer organoids from PDX and evaluate the feasibility of gene engineering using PDX-derived organoids. Materials and Methods KUCaP PDX series, which were established from castration-resistant prostate cancer patient tissues, were used to establish organoids. Eight organoids from eight models of KUCaP PDX were established and maintained for at least three passages. Organoids were reinjected into immunodeficient mice to establish organoid-derived xenograft (ODX) with 100% efficacy. We compared histopathological and molecular characteristics of primary patient tumors, PDX tumors, organoids, and ODX tumors. We conducted whole exome sequencing and RNA sequencing to assess the genomic profiles across the models. Firefly luciferase was introduced into organoids to show the feasibility of genetic engineering. Results Pathological findings were similar across the models derived from the same patient. WES analysis revealed most of the mutations detected in the matched patient tumors and organoids. Hierarchical clustering of RNA sequencing showed that the primary tumors, PDX tumors, organoids, and ODX tumors derived from the same patient cluster, which indicates that even after clonal selection during the establishment of organoids, the basic molecular feature of the model is maintained (Figure). Firefly luciferase was successfully introduced in seven out of seven tested organoids and imaged the ODX tumors from these organoids in vivo. Conclusions Organoids were established with a high success rate using PDX. Firefly luciferase was successfully introduced, showed the feasibility of genetic engineering using prostate cancer organoids. Matched PDX and organoids of PCa recapitulate the basic molecular features of primary human PCa. PDX-derived organoids of PCa, which enables genetic engineering of human-derived cellular models, which could be a valuable tool to enhance PCa research.

Citation Format: Takuro Sunada, Takayuki Goto, Takayuki Sumiyoshi, Akamatsu Shusuke, Takashi Kobayashi. Prostate cancer organoids from KUCaP patient-derived xenograft(PDX) series enables us to genetic engineering human-derived cellular models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 171.
Abstract 1712: Targeting asparagine dependency as a therapy for TP53-mutated castration-resistant prostate cancer
Authors: Yoo Young A
Published: June 2023

Background: The TP53 tumor suppressor is frequently altered in lethal, castration-resistant prostate cancer (CRPC). However, to date there are no effective treatments that specifically target TP53 gain-of-function (GOF) mutants. Thus, there is an urgent need to identify and target vulnerabilities that perpetuate disease progression and treatment resistance resulting from TP53 GOF mutants.

Methods: We performed RNA sequencing (RNA-seq) and liquid chromatography-mass spectrometry (LC-MS)-based metabolomics on GEM and PDX CRPC models with or without TP53 GOF mutations. Our findings were validated using genetic and pharmacologic approaches in relevant in vitro and in vivo models. We assessed the effect of L-asparaginase (ASNase) or/and glutaminase (GLS) inhibitor CB-839 in cell lines, organoids and PDX models. Tumors were characterized by histology and immunohistochemistry for relevant markers.

Results: Using transcriptomic and metabolomic analyses, we show here that TP53-mutated prostate cancer (PCa) exhibits an increased dependency on asparagine and overexpresses asparagine synthetase (ASNS, the enzyme catalyzing the synthesis of asparagine). Mechanistically, TP53 GOF mutations transcriptionally activate ASNS expression, directly as well as via ATF4, driving de novo asparagine biosynthesis to support CRPC growth. TP53-mutated CRPC cells are sensitive to asparagine restriction by knockdown of ASNS and L-asparaginase treatment to deplete the intracellular and extracellular sources of asparagine respectively. This effect was rescued by asparagine addition. Notably, pharmacological inhibition of intracellular asparagine biosynthesis using a glutaminase (GLS) inhibitor and depletion of extracellular asparagine with L-asparaginase significantly reduced asparagine production and effectively impaired CRPC growth.

Conclusions: This study highlights the significance of ASNS-mediated metabolic adaptation as a synthetic vulnerability in CRPC with TP53 GOF mutations, providing a rationale for co-targeting intracellular and extracellular asparagine production to treat these lethal prostate cancers.

Citation Format: Young A Yoo. Targeting asparagine dependency as a therapy for TP53-mutated castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1712.
Abstract 1720: Examining mechanistic underpinnings of chemoresistance in triple negative breast cancer
Authors: Wu Qitong , Siddharth Sumit , Verma Deepak , Parida Sheetal , Sharma Dipali
Published: June 2023

Background: Triple negative breast cancer (TNBC) is not only the most aggressive subtype of breast cancer, but it does also not have many targeted therapeutic options due to the lack of hormone receptor expression and enrichment of HER2. TNBCs are also more prone to the development of chemoresistance and metastatic progression, which are the main obstacles to reducing TNBC-related mortality. The objective of this study is to identify targetable key node (s) that contribute to the development of chemoresistance in TNBC.

Method: Differentially expressed genes in TNBC patients with or without relapse were analyzed to select functionally important genes in chemoresistant TNBC. TRIM29 was selected based on survival analysis. Carboplatin-resistant TNBC cells were established to explore the phenotypic and molecular differences. Various growth and migration assays were used to explore the phenotype of chemoresistant TNBC cells. The expression of TRIM29 and related pathways were assessed by immunoblotting and immunofluorescence analysis. Cells with TRIM29-knockout (KO) were developed by the CRISPR system.

Result: Chemoresistant TNBC cells overexpress TRIM29. Exhibiting the functional importance, overexpression of TRIM29 in MDAMB231 confers resistance to carboplatin. A stable knockout of TRIM29 in carboplatin-resistant cells results in an improved response to carboplatin. The breast tumor xenograft model revealed that TRIM29-KO in chemoresistant TNBC formed significantly less dense tumor in mice compared to control. Mechanistically, an enhanced expression of β-catenin was seen in chemoresistant as well as TRIM29-overexpressing cells. TRIM29-KO in carboplatin-resistant cell line results in a drastic decrease in β-catenin level. Combination treatment of carboplatin and β-catenin-inhibitor resulted in an enhanced growth inhibition in chemoresistant TNBC cells. RNA-Seq analysis revealed that there are 24 candidate genes which are overexpressed in chemoresistant TNBC cells but downregulated in TRIM29-KO chemoresistant cells, thereby demanding further investigation.

Conclusion: Our findings implicate TRIM29 enrichment as an important node in chemoresistant TNBC that may concomitantly modulate β-catenin as downstream oncogenic effectors.

Citation Format: Qitong Wu, Sumit Siddharth, Deepak Verma, Sheetal Parida, Dipali Sharma. Examining mechanistic underpinnings of chemoresistance in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1720.
Abstract 1840: Methylmalonic acid accumulation suppresses CD8+ T cell activation and contributes to age-induced immunosuppression
Authors: Tejero Joanne D. , Drapela Stanislav , Fernandez-Garcia Juan , Fendt Sarah-Maria , Ilter Didem , Gomes Ana P.
Published: June 2023

Deregulation of cytotoxic CD8+ T cells is a feature of the aging process with important consequences for anti-tumor immunity and consequently affects tumor initiation, progression, and response to immunotherapies. However, our understanding of how the aging process affects CD8+ T cells remains incomplete. Vitamin B12 deficiency is also a feature of old age caused by a well-recognized decrease in vitamin absorption. Interestingly, clinical studies have reported a significant decline in CD8+ T cells in individuals with vitamin B12 deficiency, suggesting that vitamin B12 levels might be a mechanistic link between aging and deregulation of CD8+ T cells. An important and often overlooked consequence of vitamin B12 deficiency is an elevation of methylmalonic acid (MMA) in circulation. Our laboratory has previously established a critical role for age-induced MMA in promoting progression into metastatic disease of breast and lung cancers, which coincided with a robust immunosuppressive signature. Here, we demonstrate that exposure of CD8+ T cells to MMA or culture of CD8+ T cells in the absence of vitamin B12 reduces T cell activation, thereby decreasing T cell numbers and effector function. Conversely, supplementation of vitamin B12 on CD8+ T cells isolated from old mice enhances their ability to activate and increases T cell numbers. Evaluation of the effects of MMA on the CD8+ T cells metabolome revealed a decrease in antioxidant levels, including a pronounced reduction in vitamin C and reduced glutathione, which were accompanied by an increase in both superoxide and hydrogen peroxide, suggesting that an increase in reactive oxygen species might be the link between MMA and CD8+ T cell dysfunction. Together, these results put forward the idea that MMA is an important regulator of immune cell function and that supplementation of vitamin B12 in older people and individuals diagnosed with vitamin B12 deficiency may function as an important strategy to improve immune cytotoxic responses and thereby increase anti-cancer immunosurveillance and the effectiveness of certain immunotherapies.

Citation Format: Joanne D. Tejero, Stanislav Drapela, Juan Fernandez-Garcia, Sarah-Maria Fendt, Didem Ilter, Ana P. Gomes. Methylmalonic acid accumulation suppresses CD8+ T cell activation and contributes to age-induced immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1840.
Abstract 1853: Microparticle-delivered Cxcl9 prolongs Braf inhibitor efficacy in melanoma
Authors: Romano Gabriele , Paradiso Francesca , Li Peng , Shukla3 Pooja , Barger Lindsay , Naggar Olivia El , Miller John P. , Liang Roger J. , Helms Timothy L. , Wargo Jennifer A. , Taraballi Francesca , Costello James C. , Kwong Lawrence N.
Published: June 2023

Patients with BRAF-mutant melanoma show significant responses to combined BRAF and MEK inhibition, but most patients relapse within 2 years. A major reservoir for such drug resistance is minimal residual disease (MRD), which is comprised of drug-tolerant tumor cells laying in a dormant state. Towards exploiting potential therapeutic vulnerabilities of MRD, we established a genetically engineered mouse model of BrafV600E-driven melanoma MRD wherein genetic BrafV600E extinction leads to strong but incomplete tumor regression. Transcriptional time-course analysis of tumors after BrafV600E extinction revealed that after an initial surge of immune activation, tumors later became immunologically "cold" after MRD establishment. Computational analysis identified candidate T-cell recruiting chemokines that may be central players in the process, being strongly upregulated initially and then steeply decreasing as the immune response faded. As a result, we hypothesized that sustaining the chemokine signaling could impair MRD maintenance through increased recruitment of effector T-cells. We show that intratumoral administration of recombinant Cxcl9, either naked or loaded in microparticles, significantly impaired the relapse of MRD in BRAF-inhibited tumors, including several complete pathological responses after the triple combination of microparticle-delivered rCxcl9 with BRAF and MEK-inhibition. Our experiments constitute a proof of concept that chemokine-based microparticle delivery systems are a potential strategy to forestall tumor relapse and thus improve the clinical success of frontline treatment methods.

Citation Format: Gabriele Romano, Francesca Paradiso, Peng Li, Pooja Shukla3, Lindsay Barger, Olivia El Naggar, John P. Miller, Roger J. Liang, Timothy L. Helms, Jennifer A. Wargo, Francesca Taraballi, James C. Costello, Lawrence N. Kwong. Microparticle-delivered Cxcl9 prolongs Braf inhibitor efficacy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1853.
Abstract 2017: A liquid biopsy approach to gastric cancer reveals a signature reflective of epithelial to mesenchymal transition
Authors: Sater Ali H. Abdel , Chen Yihui , Fahrmann Johannes F. , Irajizad Ehsan , Cai Yining , Hsiao Fuchung , Vykoukal Jody V. , Katayama Hiroyuki , Léon-Letelier Ricardo A. , Dou Rongzhang , Ostrin Edwin J. , Pizzi Melissa , Yamashita Kohei , Da Silva Matheus Sewastjanow , Song Shumei , Ajani Jaffer , Hanash Samir
Published: June 2023

Comprehensive proteomic profiling of plasmas collected before and after neo-adjuvant treatment for gastric cancer (GC) yielded 522 proteins exhibiting higher pre-treatment levels compared to healthy controls and among them, 345 proteins decreased after the treatment. Increased levels of cytoskeleton-related proteins were manifest in the plasmas of GC patients. Cytoskeletal proteins are known to play integral roles in modulating epithelial plasticity by altering mechanical strength and structural design that is critical for cancer cell motility, epithelial to mesenchymal transition (EMT), metastasis, and invasion. Ingenuity Pathway Analysis of highly expressed circulating proteins identified key oncogenic upstream transcriptional regulators and proteins that promote EMT, including KRAS, MYC and TGFB. Additionally, pathways involved in remodeling of epithelial adherens junctions, actin cytoskeleton signaling and VEGF signaling that also play a key role in EMT were upregulated. Additional datasets from proteomic profiling of multiple (N=6) GC cell lines were intersected with GC patient plasma profiles provided supporting evidence of a cancer cell origin of circulating cytoskeletal features. ACTC1, ACTN1, ACTR3, MYH9, TUBA1A, TUBA4A and TLN1 were highly expressed in both conditioned media released from GC cell lines and plasma samples of GC patients. Our findings are indicative of a blood-based signature of cytoskeletal proteins that is reflective of EMT in the context of gastric cancer.

Citation Format: Ali H. Abdel Sater, Yihui Chen, Johannes F. Fahrmann, Ehsan Irajizad, Yining Cai, Fuchung Hsiao, Jody V. Vykoukal, Hiroyuki Katayama, Ricardo A. Léon-Letelier, Rongzhang Dou, Edwin J. Ostrin, Melissa Pizzi, Kohei Yamashita, Matheus Sewastjanow Da Silva, Shumei Song, Jaffer Ajani, Samir Hanash. A liquid biopsy approach to gastric cancer reveals a signature reflective of epithelial to mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2017.
Abstract 2022: What can we learn from phosphoproteomics compared to transcriptomics in DNA damage and other processes
Authors: Arad Gali , Pevzner Kirill , Seger Eran , Geiger Tamar
Published: June 2023

The advancement of sequencing technologies made unbiased whole genome and transcriptome studies ubiquitous in the cancer research field. However, these omics analysis methods do not describe post-transcriptional and post-translational levels. With the recent availability of large scale multi-omics datasets, we set out to study the correlation between phosphorylations, protein expression and gene expression. The goal of this analysis is to highlight the cases in which systematic characterization of the whole proteome and phosphoproteome can uncover insights that are undetected with RNA sequencing.

The analysis described below applies RNA-protein-phosphorylation comparisons based on clinical cancer datasets. We make use of the large amount of data generated by the Clinical Proteomic Tumor Analysis Consortium (CPTAC), which generates unique phosphorylations, protein and mRNA expression data from the exact same samples.

Correlation analysis between the phosphorylation-protein-RNA profiles highlights multiple differences among cancer types and functional pathways. We specifically focus on the various DNA damage response pathways which are mediated by kinase activity, and as expected, show modest mRNA-protein correlations. The protein-RNA discordance is further demonstrated when using an unsupervised clustering of the data based on protein or RNA. This analysis showed substantial differences in tumor classification and the cellular processes that differentiate between clusters.

Altogether, this study demonstrates the opportunity presented by applying proteomics and phosphoproteomics techniques to study multiple systems that are at play in cancer biology, highlighting DNA damage response as a striking example.

Citation Format: Gali Arad, Kirill Pevzner, Eran Seger, Tamar Geiger. What can we learn from phosphoproteomics compared to transcriptomics in DNA damage and other processes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2022.
Abstract 2090: Comprehensive interrogation of gene lists from genome-scale cancer screens with oncoEnrichR
Authors: Nakken Sigve , Gundersen Sveinung , Bernal Fabian L. , Polychronopoulos Dimitris , Hovig Eivind , Wesche Jørgen
Published: June 2023

Genome-scale screening experiments in cancer produce long lists of candidate genes that require extensive interpretation for biological insight and prioritization for follow-up studies. Interrogation of gene lists frequently represents a significant and time-consuming undertaking, in which experimental biologists typically combine results from a variety of bioinformatics resources in an attempt to portray and understand cancer relevance. As a means to simplify and strengthen the support for this endeavor, we have developed oncoEnrichR, a flexible bioinformatics tool that allows cancer researchers to comprehensively interrogate a given gene list along multiple facets of cancer relevance. oncoEnrichR differs from general gene set analysis frameworks through the integration of an extensive set of prior knowledge specifically relevant for cancer, including ranked gene-tumor type associations, literature-supported proto-oncogene and tumor suppressor gene annotations, target druggability data, regulatory interactions, synthetic lethality predictions, as well as prognostic associations, gene aberrations, and co-expression patterns across tumor types. The software produces a structured and user-friendly analysis report as its main output, where versions of all underlying data resources are explicitly logged, the latter being a critical component for reproducible science. We demonstrate the usefulness of oncoEnrichR through interrogation of two candidate lists from proteomic and CRISPR screens. oncoEnrichR is freely available as a web-based workflow hosted by the Galaxy platform (https://oncotools.elixir.no), and can also be accessed as a stand-alone R package (https://github.com/sigven/oncoEnrichR).

Citation Format: Sigve Nakken, Sveinung Gundersen, Fabian L. Bernal, Dimitris Polychronopoulos, Eivind Hovig, Jørgen Wesche. Comprehensive interrogation of gene lists from genome-scale cancer screens with oncoEnrichR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2090.
Abstract 2110: Reprogramming of DNA methylation in NAFLD progression
Authors: Go Hanyong , Kim Seon-Kyu , Kim Mirang
Published: June 2023

NAFLD has several stages in its progression that can lead to non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), cirrhosis, and even hepatocellular carcinoma (HCC). Whereas NAFL is a simple steatosis, NASH is characterized by cell injury, inflammation, and hepatocyte ballooning that may further progress to fibrosis, cirrhosis, and HCC. DNA methylation is a relatively stable epigenetic mechanism that can regulate gene expression patterns to establish cell identity. Here we investigated liver DNA methylation in individuals with simple steatosis and NASH, and further tested if these alterations were associated with clinical phenotypes. NAFLD is associated with abnormal DNA methylation, Advanced NAFLD is associated with hypomethylation of genes involved in TNF receptor signaling and apoptosis, and hypermethylation of genes associated with lipid metabolism, digestion and transport. We suggest that reprogramming of DNA methylation contributes to NAFLD progression and aberrant DNA methylation could be a predictive biomarker of NASH associated HCC.

Citation Format: Hanyong Go, Seon-Kyu Kim, Mirang Kim. Reprogramming of DNA methylation in NAFLD progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2110.
Abstract 221: Development of a rapid, highly sensitive cell-free RNA NGS assay for the detection of gene fusion and splicing variants in cancers
Authors: liu fang , sun xiangying , dong jianrong , zhou kemin , huang yong , jia shidong , du pan , xiang Binggang , luo shujun
Published: June 2023

Background: Utility of cell-free nucleic acids (cf-NAs) for early detection and surveillance of tumor represents a new path in personalized medicine. Gene fusions, which usually arise from chromosomal rearrangement, play pivotal roles in numerous human malignancies and have been exploited for disease diagnosis and therapeutics. RNA sequencing, which is not influenced by the complication of introns, is considered superior to DNA sequencing for the identification of fusion genes and splicing variants, and has emerged as a critical tool in cancer liquid biopsy.

Methods: We developed a novel approach of preparing strand-specific RNA-Seq libraries. The cDNA produced from the cell-free RNA (cfRNA) samples were labelled with unique molecular identifiers (UMI) during library construction. The libraries were enriched using hybrid capture and the sequencing data were analyzed using Predicine in-house analysis pipeline. To assess the performance of the newly developed cfRNA assay, we compared it with a variety of commercial RNA library kits.

Results: The new RNA library construction workflow was streamlined, and it reduces the library preparation time from 2 days to 6 hours with less than 1 h of hands-on time. The yield of the library prepared by the new method was higher than those of the commonly used commercial kits on the market. Sequencing analysis and Droplet Digital PCR (ddPCR) measurement both revealed that our assay was more sensitive in detecting gene fusions and splicing variants in cfRNA than other commercial kits. The assay can reliably detect fusion/splicing variants down to the level of <10 copies of input cfRNA with high specificity.

Conclusions: We developed a new RNA library preparation method that produced strand-specific libraries from low input quantities of cfRNA in a fast, simple, and robust manner. This RNA-Seq assay has demonstrated superior performance in known fusion detection and novel fusion discovery in cfRNA.

Citation Format: fang liu, xiangying sun, jianrong dong, kemin zhou, yong huang, shidong jia, pan du, Binggang xiang, shujun luo. Development of a rapid, highly sensitive cell-free RNA NGS assay for the detection of gene fusion and splicing variants in cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 221.
Abstract 2213: Longitudinal tracking of circulating rare events in the liquid biopsy of stage III-IV NSCLC patients during treatment
Authors: Bai Lily , Courcoubetis George , Nieva Jorge , Mason Jeremy , Kuhn Peter , Shishido Stephanie
Published: June 2023

Lung cancer is the most common cause of cancer-related death in the United States. Non Small Cell Lung Cancer (NSCLC) is the most prevalent subtype of lung cancer diagnoses, with significantly worse survival rates as clinical staging progresses. The purpose of this study was to determine the prognostic utility of longitudinal peripheral liquid biopsy as a treatment monitoring tool in stage III-IV NSCLC patients throughout treatment. Liquid biopsy represents a way for physicians to non-invasively continuously monitor and understand tumor progression through analyzing serial patient blood samples. This allows for real-time, in depth analysis of patient status throughout the treatment process. This study used the third generation, enrichment-free High-Definition Single Cell Assay (HDSCA3.0) workflow to detect and characterize rare events in the 48 peripheral blood samples collected from a cohort of 10 stage III-IV NSCLC patients undergoing various treatments. This workflow uses 4 fluorescent channels corresponding to 5 biomarkers. While circulating tumor cells (CTCs) have been shown to be effective as a prognostic tool for measuring treatment efficacy and patient response to treatment, we have additionally identified 6 additional rare cell phenotypes with variable cytokeratin expression, and 3 types of oncosomes. We have established a liquid biopsy profile that is unique to NSCLC as compared to 50 normal donors. Within this cohort, we found significantly higher amounts of rare cells and oncosomes in patient cohorts as compared to normal donors, highlighting the potential utility of both cellular and oncosome subtypes in disease monitoring. In addition, these rare events differ drastically in morphological parameters and channel expression, and also display temporal heterogeneity. Through the analysis of peripheral blood samples across time and treatment methodologies, we observed significant association between liquid biopsy analytes and progression-free and overall survival, supporting the utility of liquid biopsy as a clinical tool. Understanding the dynamics behind these rare groups throughout the courses of treatment may help improve prognostic utility of liquid biopsy.

Citation Format: Lily Bai, George Courcoubetis, Jorge Nieva, Jeremy Mason, Peter Kuhn, Stephanie Shishido. Longitudinal tracking of circulating rare events in the liquid biopsy of stage III-IV NSCLC patients during treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2213.
Abstract 2217: Novel risk stratification utilizing NanoString for HPV+ head and neck cancer patients
Authors: Kim Sulgi , Schrank Travis , Kothari Aditi , Rehmani Hina , Tasoulas Jason , Li Xue , Yarbrough Wendell , Isaeva Natalia
Published: June 2023

Incidence of human papillomavirus positive (HPV+) head and neck squamous cell carcinoma (HNSCC) has dramatically increased in the U.S. surpassing cervical cancer since 2012. Among HNSCC, HPV+ HNSCC patients have a favorable prognosis; however, approximately 20-30% suffer recurrence. Given the morbidity of current therapy, therapeutic de-escalation for HPV+ HNSCC is being explored in many clinical trials but is hampered by absence of accurate molecular classifiers to identify tumors at high and low risk of recurrence. Being able to risk stratify patients can help with therapeutic decisions, maintain high cure rates and decrease treatment associated complications. We assemble a NanoString panel to capture our recently developed NF-κB RNA signature that can distinguish patients requiring aggressive therapy from patients who are appropriate for reduced intensity treatment. Our custom gene set can select HPV+ head and neck cancer patients that may benefit from therapy de-escalation. Several advantages of using a NanoString technology, including smaller amounts of patient samples and less processing time that allow real-time patient-specific results. This makes our Nanostring panel suitable to be used in clinic to identify HPV+ head and neck cancer patients for therapeutic de-escalation without compromising their survival.

Citation Format: Sulgi Kim, Travis Schrank, Aditi Kothari, Hina Rehmani, Jason Tasoulas, Xue Li, Wendell Yarbrough, Natalia Isaeva. Novel risk stratification utilizing NanoString for HPV+ head and neck cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2217.
Abstract 2287: Circulating extracellular vesicles from patients with advanced pancreatic cancer contain low levels of tumor-derived DNA
Authors: Lapin Morten , Tjensvoll Kjersti , Nedrebø Karoline , Taksdal Eline , Janssen Hans , Gilje Bjørnar , Nordgard Oddmund
Published: June 2023

Purpose: Extracellular vesicles (EVs) have been shown to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform circulating cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated whether EVs could be a potential source of tumor-derived DNA in patients with advanced pancreatic cancer.

Methods: evDNA from both DNase-treated and untreated circulating EVs, enriched from patients with advanced pancreatic cancer (n=10), was analyzed by fluorometry and digital droplet PCR to determine the level of evDNA inside and outside (surface-bound) the EVs. To also determine if choice of methodology affected the results, EVs were isolated using four different small EV isolation methods (differential ultracentrifugation, size-exclusion chromatography, affinity purification and precipitation) and differential centrifugation for isolation of large EVs. Western blotting, dynamic light scattering and transmission electron microscopy confirmed the enrichment of EVs.

Results: Our results indicated that EVs enriched from patient blood samples contained significantly lower amounts of DNA compared to cfDNA (p < 0.001); >50-fold lower for untreated sEVs and >1000-fold lower for DNase-treated EVs. Thus, most of the detected vesicle-associated DNA seemed to be located outside of the vesicles. Further, tumor-derived DNA in EVs was barely detectable and the tumor-derived fraction in all evDNA samples was similar to that of cfDNA.

Conclusion: Our results demonstrated that evDNA do not add any extra information compared with cfDNA as a source of tumor-derived DNA, at least not in patients with advanced pancreatic cancer.

Citation Format: Morten Lapin, Kjersti Tjensvoll, Karoline Nedrebø, Eline Taksdal, Hans Janssen, Bjørnar Gilje, Oddmund Nordgard. Circulating extracellular vesicles from patients with advanced pancreatic cancer contain low levels of tumor-derived DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2287.
Abstract 2656: Reversible chemical modification of antibodies: A complementary approach to tuning FcγR binding that maintains anti-tumor activity while mitigating peripheral immune activation
Authors: Moquist Philip N. , Leiske Chris I. , Bindman Noah A. , Zhang Xinqun , Duncan Nicole , Zeng Weiping , Wo Serena W. , Wong Abbie , Henderson Clark M. , Crowder Karalyne , Neff-LaFord Haley D. , Sussman Django , Gardai Shyra J. , Levengood Matthew R.
Published: June 2023

Modification of effector function has proven to be an effective modality for optimizing activity and tolerability of therapeutic antibodies. Currently available methods to modulate effector function include the introduction of point mutations in the Fc region and glycan engineering of the antibody. Here we present an alternative and complementary method of tuning effector function utilizing a conjugation-based approach. This methodology uses conjugation of polyethylene glycol (PEG) to native cysteines of an antibody to impair FcγR binding of antibodies to innate immune effector cells. Utilizing maleimide or disulfide conjugation techniques, attenuated effector function can be either permanent or restored over time through a de-conjugation process. Impacts of PEGylation on FcγR binding, signaling, and restoration of function were assessed in vitro and in vivo. As a proof-of-concept, the lead technology was applied to an agonist CD40 antibody, which resulted in significant reductions in systemic cytokine production in hCD40 mice and non-human primates, while demonstrating retained efficacy and improved pharmacokinetics. Additionally, we combined the conjugation technology with glycan engineering and FcγR enhancing point mutations to impart unique effector function profiles to clinical antibodies. This simple, modular approach can be rapidly applied to existing antibodies to reduce immune-driven toxicities, such as infusion reactions, and optimize effector function activity.

Citation Format: Philip N. Moquist, Chris I. Leiske, Noah A. Bindman, Xinqun Zhang, Nicole Duncan, Weiping Zeng, Serena W. Wo, Abbie Wong, Clark M. Henderson, Karalyne Crowder, Haley D. Neff-LaFord, Django Sussman, Shyra J. Gardai, Matthew R. Levengood. Reversible chemical modification of antibodies: A complementary approach to tuning FcγR binding that maintains anti-tumor activity while mitigating peripheral immune activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2656.
Abstract 2678: Combination therapy with cabozantinib and cannabidiol enhances the apoptotic cell death via phosphorylation ofTP53 regulated by ER-stress in hepatocellular carcinoma
Authors: Jeon Youngsic , Kim Taejung , Kwon Hyukjoon , Ham Jungyeob , Kim Young-Joo
Published: June 2023

Cannabidiol (CBD), a primary constituent of hemp and cannabis, has pharmacologically broad properties for a variety of diseases including cancer. Cabozantinib (CBZ), a potent multikinase inhibitor, has been approved for use in patients with advanced hepatocellular carcinoma (HCC). Recently, research is increasing for combination therapy using CBZ for better improved efficacy and safety for the patients. Here, we investigated the effect of the combination therapy with CBZ and CBD in HCC cells. CBD enhances the sensitivity of CBZ against HCC cells by increasing cytotoxicity and apoptosis. Especially, analysis of phospho-kinase array demonstrated that the apoptotic effect of combination treatment with CBZ and CBD was significantly related phosphorylation of TP53 regulated by endoplasmic reticulum stress (ER-stress) compared to other kinases. Inhibition of TP53 expression and ER-stress abolished the apoptotic effect of combination treatment with CBZ and CBD, revealing no expression changes in Bax, Bcl-2, cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9. On the other hand, we found that the effect of combination treatment with CBZ and CBD was not associated with cannabinoid receptor 1 (CNR1) and CNR2 receptors signaling pathway. Our findings suggest that combination therapy with CBZ and CBD provides enhanced therapeutic efficacy against HCC.

Citation Format: Youngsic Jeon, Taejung Kim, Hyukjoon Kwon, Jungyeob Ham, Young-Joo Kim. Combination therapy with cabozantinib and cannabidiol enhances the apoptotic cell death via phosphorylation ofTP53 regulated by ER-stress in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2678.
Abstract 2855: Epithelial-mesenchymal transition profile after cisplatin-resistance induction in OSCC cell line
Authors: De Lima Dieila Giomo , Borgato Gabriell Bonifácio , Guimarães Gustavo Narvaes , Bastos Débora Campanella , Coletta Ricardo Della , De Souza Ana Paula
Published: June 2023

Oral cancer is among the most common types of cancer in humans. Chemotherapy, mainly based on cisplatin, is utilized in the treatment of advanced cases. However, a loss of chemotherapy effectiveness is frequent and can be related to both inherent or acquired resistance to the chemotherapy agent, resulting in high relapse rates. Epithelial-mesenchymal transition (EMT) plays an important role in different stages of cancer, and it is correlated to resistance to therapy, but the process is still unclear. The purpose of this study is to explore EMT markers in a cisplatin resistant-based model. After determination of IC50, SCC9 cells were intermittently exposed to stepwise increasing concentrations of cisplatin, and Resistance Index (RI) was calculated. Population Doubling (PD) and Population Doubling Time (PDT) were calculated counting the number of viable cells using Trypan Blue method. Scratch assay was performed to investigate the impact on cellular migration. EMT-correlated markers were accessed by RT-qPCR and western blot. After 4 intermittent exposures to cisplatin, we obtained different clones with cisplatin resistance level, SCC9/CPR5 (intermediate cisplatin resistance) and SCC9/CPR11 (final cisplatin resistance) the SCC9/CPR5 clone was obtained showing a mixed morphology between polygonal and fusiform. The complete fusiform morphology resembling mesenchymal cells was seen in SCC9/CPR11, which demonstrated 2-fold of cisplatin resistance in comparison to parental cell line. SCC9/CPR11 showed a difference of less than 1.16 PD and more than 15.5 hours PDT in relation to SCC9. Interestingly, the migratory profile was increased in SCC9/CPR5. Snail1 and Vimentin mRNA levels and protein expression were increased at SCC9/CPR11, but surprisingly E-cadherin was overexpressed but not in protein level. Others markers such as Zeb1, N-cadherin, Vimentin, Snail2 and Twist showed an increase in mRNA levels only in SCC-9-CPR11, however only Twist exhibited high protein expression. Our study showed the establishment of cisplatin resistant SCC9 cells in different moments in relation to a resistance level based on the main two protein markers of EMT, Snail1 and Vimentin. Our data need to be further validated in larger well characterized techniques and we further envision investigating the epigenetic in DNA damage pathways correlating with cisplatin resistance and EMT status.

Citation Format: Dieila Giomo De Lima, Gabriell Bonifácio Borgato, Gustavo Narvaes Guimarães, Débora Campanella Bastos, Ricardo Della Coletta, Ana Paula De Souza. Epithelial-mesenchymal transition profile after cisplatin-resistance induction in OSCC cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2855.
Abstract 2858: Radiotherapy and NK cell-based immunotherapy: An evolutionary double-bind
Authors: Luddy Kimberly , West Jeffery , Robertson-Tessi Mark , Desai Bina , Bursell Taylor M. , Barrett Sarah , Brown Joel , O'Sullivan Jacintha , Gatenby Robert , Marignol Laure , Anderson Alexander R. , O'Farrelly Cliona
Published: June 2023

Evolution-informed therapies exploit the ecological and evolutionary consequences of drug resistance to inhibit the expansion of treatment resistant populations and prolong time to progression. One strategy, termed an evolutionary double-bind, uses an initial therapy to elicit a specific adaptive mechanism that is then selectively targeted by a follow-on therapy. Here we examine combining radiation therapy followed by immunotherapy as a double bind strategy. Radiotherapy (RT) induces lethal double-strand DNA breaks in tumor cells and radiosensitivity is governed by the cells’ DNA repair capabilities. Radioresistant cancer cells upregulate DNA damage response pathways. However, successful adaptations to RT and other DNA damaging agents can increase natural killer (NK) cell ligand expression on tumor cells increasing their vulnerability to the immune system.

We conducted a multidisciplinary investigation of this potential evolutionary double bind using an isogenic cell line model of radiation resistance derived from an androgen-independent human prostate carcinoma cell line (22Rv1). We demonstrate that RT-resistant cells maintain higher expression of NK cell ligands and have a 2-fold increase in sensitivity to NK cell mediated killing. Furthermore, we show that preferential targeting of the RT-resistant population by NK cells is preserved in competition assays with varying initial population frequencies. Scope of treatment responses are dependent on population frequencies in our in vitro analysis.

Mathematical modeling quantified three key elements of a double-bind: cost of resistance, inter-specific competition between RT-sensitive and RT-resistant lines, and preferential targeting of RT-resistant lines by NK cells. We conclude that RT followed by immunotherapy produces an evolutionary double bind that can be exploited in heterogenous tumors to limit RT resistance. Furthermore, model simulations predict that extinction of both populations is achievable by sequential RT and NK cell therapy.

Citation Format: Kimberly Luddy, Jeffery West, Mark Robertson-Tessi, Bina Desai, Taylor M. Bursell, Sarah Barrett, Joel Brown, Jacintha O'Sullivan, Robert Gatenby, Laure Marignol, Alexander R. Anderson, Cliona O'Farrelly. Radiotherapy and NK cell-based immunotherapy: An evolutionary double-bind [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2858.
Abstract 3088: Identification of a novel series of MTAP-selective PRMT5 inhibitors, and first disclosure of AZ-PRMT5i-1
Authors: Smith James M. , Barlaam Bernard , Beattie David , Cumming Iain , Robb Graeme , Wilson David , Gianni Davide , Srinivasan Bharath , Stubbs Chris , Debreczeni Judit , Vazquez-Chantada Mercedes , Chiarparin Elisabetta , Lynch James , Dean Emma
Published: June 2023

Background: PRMT5, a type 2 arginine methyltransferase, has a critical role in regulating cell growth and survival in cancer. Despite this therapeutic potential, there is growing evidence that the first generation of clinical PRMT5 inhibitors may not be able to achieve high clinical efficacy due to PRMT5-driven (e.g. hematological) toxicities.

Aims: In order to pharmacologically exploit the MTAP ‘collateral vulnerability’ mechanism for the potential of increased therapeutic index and clinical efficacy, next-generation PRMT5 inhibitors are required to bind PRMT5 in a cooperative manner with natural metabolite methylthioadenosine (MTA). Whilst MTAP-selective PRMT5 inhibitors have recently been reported,1,2,3 there is continued interest in identifying new and structurally diverse in vivo-quality compounds to further investigate this therapeutic opportunity.

Results: To identify novel inhibitors of PRMT5 suitable for the MTAP line of sight, our hit finding strategy used a ‘biased’ biochemical high throughput screen of a 2.4M compounds collection. MTA-cooperative hits were validated using techniques including SPR and Xray crystallography. One series identified from this screen was extensively optimized using techniques including structure-based design to improve potency, MTAP selectivity and DMPK properties. Through these optimizations, we have identified AZ-PRMT5i-1, which shows potent in vitro cellular inhibition of PRMT5, over 50-fold MTAP selectivity margin, excellent selectivity against a broad panel of methyltransferases, and suitable oral PK properties.

Conclusion: AZ-PRMT5i-1 is a structurally novel and potent PRMT5 inhibitor with high MTAP-selectivity suitable for use in in vitro and in vivo PRMT5-driven models of cancer’.

Note: the structure of AZ-PRMT5i-1 will be disclosed at this meeting

PRMT5 Cell SDMA: IC50 nM (HCT116 MTAP KO/WT) SDMA MTAP selectivity (fold) LogD/Rat heps CLint/Caco2 intr. A-B HTS hit A 660/4300 6.5 2.1/34/1.6 AZ-PRMT5i-1 5.4/290 54 2.8/5.0/48

References:1. J. Med. Chem. 65, 1749-1766 (2022); 2. WO2021163344; 3. WO2022026892

Citation Format: James M. Smith, Bernard Barlaam, David Beattie, Iain Cumming, Graeme Robb, David Wilson, Davide Gianni, Bharath Srinivasan, Chris Stubbs, Judit Debreczeni, Mercedes Vazquez-Chantada, Elisabetta Chiarparin, James Lynch, Emma Dean. Identification of a novel series of MTAP-selective PRMT5 inhibitors, and first disclosure of AZ-PRMT5i-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3088.