bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5862: ABCB5 knockdown suppresses tumor-intrinsic expression of immune checkpoint regulators PD-1 and PD-L1 in hepatocellular carcinoma (HCC)
Authors: Yeung Chun Philip , Wilson Brian J. , Sasamoto Yuzuru , Waaga-Gasser Ana Maria , Karpova Svetlana , Guo Qin , Frank Natasha Y. , Frank Markus H.
Published: April 2023

ATP-binding cassette member B5 (ABCB5) identifies cancer-initiating cells (CICs) in multiple malignancies, including malignant melanoma, colorectal cancer, glioblastoma, and hepatocellular carcinoma (HCC). In human melanoma, ABCB5-positive CIC subpopulations have been shown to preferentially express the immune checkpoint molecules PD-1 and PD-L1, and tumor-intrinsic PD-1 receptor functions have been found to promote tumor growth, even in mice lacking adaptive immunity. In HCC, where ABCB5 mediates CIC chemoresistance, PD-1 expression has also recently been identified. However, whether ABCB5 regulates tumor-intrinsic PD-1 expression in HCC or other malignancies is currently unknown. Here, we employed RNA interference in Hep G2 HCC cells with high endogenous ABCB5 expression to study the potential role of ABCB5 in regulating PD-1 expression, utilizing the ABCB5 shRNA target sequence GCTGGAAAGATAGCAACTGAA for ABCB5 knockdown (KD) as described previously. Endogenous ABCB5 levels in ABCB5-KD cells were successfully reduced by 92.8% compared to controls. Investigation of the effects of ABCB5-KD on tumor-intrinsic immune checkpoint molecule expression and on expression of the HCC-CIC marker GRN revealed that ABCB5-KD significantly reduced PD-1 expression by 98.0%, and PD-L1 expression by 62.8% compared to controls. Expression of GRN was also significantly reduced in ABCB5-KD cells by 78.8% compared to controls, suggesting ABCB5 KD-dependent loss of CIC phenotype. Injection of ABCB5-KD cells in NSG mice showed 32.3% reduction in tumor size at endpoint (6 weeks) compared to controls (728.7 ± 199.3mm3 vs 1076.0 ± 450.9 mm3, mean ± SD; p<0.005, repeated measures ANOVA) and 8.53% reduction in tumor weight at endpoint (0.47 ± 0.21g vs 0.51 ± 0.21g, mean ± SD). These results provide initial evidence in an HCC cell line model system that ABCB5 functionally regulates tumor-intrinsic PD-1 and PD-L1 expression. They therefore suggest that ABCB5 blockade represents a novel therapeutic strategy for targeted inhibition of pro-tumorigenic tumor-intrinsic PD-1/PD-L1 expression, with important additional implications for immune checkpoint-targeted cancer immunotherapy.

Citation Format: Chun Philip Yeung, Brian J. Wilson, Yuzuru Sasamoto, Ana Maria Waaga-Gasser, Svetlana Karpova, Qin Guo, Natasha Y. Frank, Markus H. Frank. ABCB5 knockdown suppresses tumor-intrinsic expression of immune checkpoint regulators PD-1 and PD-L1 in hepatocellular carcinoma (HCC) [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 5862.
Abstract 5884: Targeting T regulatory cells to intercept progression from preinvasive to invasive lung adenocarcinoma using a novel KRAS-driven murine model
Authors: Martin Mitchell , Crowley Michael J. , Markowitz Geoffrey , Borczuk Alain , Zhang Chen , Yoffe Liron , Singh Arshdeep , Altorki Nasser K. , Mittal Vivek
Published: April 2023

Non-small cell lung cancer is the leading cause of cancer-related death in the US and worldwide, with a 5-year survival of ~18-20%. Understanding of the biology of early lung cancer is necessary to intercept early steps of progression. Indeed, increased utility of low-dose CT guided lung cancer screens in populations at risk have begun to detect early premalignant disease; however, little is known about the underlying biology that drives progression of preinvasive lesions to invasive adenocarcinoma. Moreover, there exists a lack of reliable animal models to assess interception strategies to target disease progression. We have developed and characterized a lung cancer cell-of-origin-specific KRAS-driven mouse model which accurately mirrors progression of human precursor lesions to invasive disease. Transcriptional analysis of preinvasive and invasive stages identified cellular and molecular alternations observed in human lesions, including upregulation of cell proliferation, extracellular matrix remodeling, and an increasingly complex immune microenvironment. Interrogation with immunofluorescence microscopy and Hyperion mass cytometry imaging showed marked reprogramming of the immune microenvironment including increased T cell infiltration and abundance of T regulatory cells (Tregs). Surprisingly, depletion of T cells impaired progression of preinvasive lesions, implicating the critical immunosuppressive roles of Tregs. Spatial transcriptomics (10X Genomics Visium) revealed unique spatial transcriptional changes in the progression from preinvasive to invasive stage disease, including specific ligand-receptor interactions highly expressed in Tregs. Genetic and pharmacological approaches using FoxP3-DTR-GFP mice and low dose cyclophosphamide have begun to define the functional role of Tregs in early premalignant disease.

This study demonstrates critical roles of immunosuppressive Tregs in driving progression of preinvasive to invasive lung cancer, and advocates for Treg targeting as a potent immunoprevention approach for patients at risk of developing lung cancer.

Citation Format: Mitchell Martin, Michael J. Crowley, Geoffrey Markowitz, Alain Borczuk, Chen Zhang, Liron Yoffe, Arshdeep Singh, Nasser K. Altorki, Vivek Mittal. Targeting T regulatory cells to intercept progression from preinvasive to invasive lung adenocarcinoma using a novel KRAS-driven murine model [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 5884.
Abstract 5898: Gut microbiota-derived metabolism of 5-fluorouracil affects drug efficacy
Authors: Chrysostomou Despoina , David Mark , Alexander James L. , Marchesi Julian R. , Kinross James M.
Published: April 2023

Introduction: Fluoropyrimidines are drugs currently used as a first-line treatment for colorectal cancer (CRC)1. The impact of gut bacteria on 5-Fluorouracil (5FU) activity remains relatively unknown. The objective of this analysis was to delineate potential bacteria-drug interactions that modify 5FU.

Methods: Tumor and tumor-adjacent healthy tissue samples from 130 colorectal adenocarcinoma patients, 55% males and 45% females, median age of 71 years old (36-91 age range), were extracted following surgical resection of the tumor. TNM classification varied across the cohort (15% pT1, 43% pT2, 35% pT3, 4% pT4). 16s rRNA gene amplicon sequencing was performed to map the microbiome composition of tumor and paired normal mucosa. 20 bacterial from the genera whose abundance changed significantly between tumor and healthy tissue were incubated with 5FU to assess drug-bacteria interactions. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was employed to identify changes in drug concentration (Wilcoxon spare rank test) as well as products of bacteria-driven drug metabolism. A metabolic activity-based assay was used to assess viability of a colorectal adenocarcinoma cell line (HCA46) following co-incubation with microbe-driven drug metabolites, aiming to determine significant effects on 5FU efficacy, assessed by 2-way ANOVA.

Results: Escherichia coli BW25113, Bifidobacterium longum, Citrobacter freundii and Enterococcus faecalis significantly modify 5FU in vitro, allowing us to conclude that bacteria can metabolise the drug (q < 0.001). Ruminococcus gnavus does not have the ability to metabolise the drug as no significant change in 5FU concentration was observed. Incubation of microbe-derived drug metabolites with HCA46 resulted in significant decrease in cell viability compared to incubation with 5FU only, indicating that bacteria-5FU interactions result in increase of drug efficacy (Figure 2).

Conclusion: This is the first report of 5FU metabolism by Escherichia coli BW25113, Bifidobacterium longum, Citrobacter freundii and Enterococcus faecalis in vitro. This study provides an insight into the potential of large and small intestinal bacteria in chemically modifying chemotherapeutic agents and therefore modulating their activity.

1. Mahipal A, Grothey A. Role of biologics in first-line treatment of colorectal cancer. J Oncol Pract. 2016.

Citation Format: Despoina Chrysostomou, Mark David, James L. Alexander, Julian R. Marchesi, James M. Kinross. Gut microbiota-derived metabolism of 5-fluorouracil affects drug efficacy [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 5898.
Abstract 5991: Development and characterization of a novel CDK4/6 inhibitor for the treatment of CDK4/6 dependent cancers
Authors: O'Brien Neil A. , McDermott Martina S. , O'Boyle Brendan M. , Hilf Justin A. , Loson Oliver , Chau Kevin , Jia Weiping , Kamranpour Naeimeh , Luo Tong , Ayala Raul , Issakhanian Shawnt , Glaspy John A. , Stoltz Brian M. , Slamon Dennis J.
Published: April 2023

CDK4/6 inhibitors give clinical benefit in the treatment of metastatic ER+/HER2- breast cancer (mBC) in combination with endocrine therapy. Although this class of inhibitor has been extensively studied in several other cancer types, CDK4/6 inhibitors have not been approved as a treatment for outside of ER+/HER2- cancer. We evaluated the clinically approved CDK4/6 inhibitors, palbociclib, ribociclib and abemaciclib—in a 500+ cell line screening platform and identified several cancers outside of breast that may have subpopulations that are sensitive to CDK4/6 inhibitors. Kinome and KiNativ analyses helped identify differences in sensitivity/selectivity that we observed between each CDK4/6 inhibitor. Comprehensive molecular profiling of the cell lines at baseline allowed us to screen for potential associations with sensitivity to these compounds. These analyses identified potential biomarkers of sensitivity in non-small cell lung carcinoma (NSCLC) and colorectal cancer (CRC) that will later be evaluated in human clinical studies. Having thoroughly studied the CDK4/6 class and discovered new opportunities for clinical development, we set out to develop a CDK4/6 inhibitor that would have the best properties of the two distinguished class members. Using our proprietary chemistry, we developed UCT-03-008, a potent CDK4/6 small molecular inhibitor with a pharmacology known to enhance therapeutic benefit in mBC. Pharmacokinetic properties of UCT-03-008 are improved over those of other CDK4/6 inhibitors. UCT-03-008 has activity in preclinical models as a monotherapy and yields in vivo efficacy in multiple cancer models outside of ER+/HER2- breast cancer, including CRC and NSCLC. In summary, UCT-03-008 is a CDK4/6 inhibitor with optimized pharmacological properties. Clinical development of this compound will be guided by the biomarker hypotheses developed here. We are currently enrolling patients for treatment with UCT-03-008 in a Phase 1 clinical trial of advanced solid tumors (NCT05103046).

Citation Format: Neil A. O'Brien, Martina S. McDermott, Brendan M. O'Boyle, Justin A. Hilf, Oliver Loson, Kevin Chau, Weiping Jia, Naeimeh Kamranpour, Tong Luo, Raul Ayala, Shawnt Issakhanian, John A. Glaspy, Brian M. Stoltz, Dennis J. Slamon. Development and characterization of a novel CDK4/6 inhibitor for the treatment of CDK4/6 dependent 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 5991.
Abstract 6004: DNA methylation diagnostics for upper urinary tract urothelial carcinoma using newly developed high-performance liquid chromatography
Authors: Fujimoto Mao , Arai Eri , Kikushima Takuya , Nishiyama Naotaka , Nakamura Tomofumi , Yamada Yuriko , Taira Hiroaki , Yotani Takuya , Kitamura Hiroshi , Kanai Yae
Published: April 2023

Backgrounds: For diagnosis of upper urinary tract urothelial carcinoma (UTUC), conventional ureteroscopic examinations are invasive, and selective urine cytology has insufficient sensitivity. We have developed a new anion-exchange column for high-performance liquid chromatography (HPLC) with electrostatic and hydrophobic properties. Both cytosine and thymine, corresponding to methylated and unmethylated cytosine after bisulfite modification, respectively, are captured by electrostatic interaction and then discriminated from each other by their hydrophobic interactions. This method is expected to be very suitable for quantification of DNA methylation levels of cancer cells in urine samples with contamination of various cell lineages.

Purpose: The aim of this study was to establish a less invasive and more accurate method for UTUC diagnosis based on DNA methylation quantification by our newly developed HPLC.

Methods: Urine samples were collected by urinary catheterization during surgery for 218 urological cancer patients. Our previous studies have identified 4 CpG sites showing differences in DNA methylation levels between non-cancerous urothelium and UTUC based on genome-wide DNA methylation analysis using tissue specimens. In the present study, DNA methylation levels of these marker CpG sites were quantified by HPLC using the urine samples.

Results: The thresholds have been set for the retention time by HPLC and the area under the chromatogram. Diagnostic criteria have been established to distinguish cases with current UTUC from non-urothelial cancer cases, bladder cancer cases and post-urothelial cancer treatment cases by combining marker CpG sites. The sensitivity and specificity were 90.9% and 87.0% respectively.

Conclusions: Since HPLC-based DNA methylation diagnostic system was able to discriminate UTUC cells from blood cells and other contaminating cells in urine samples, application of our criteria will ultimately allow non-invasive diagnosis of UTUC in the clinical setting. It may be possible to at least partially replace highly invasive procedures such as ureteroscopy and to enable early initiation of treatment for UTUC resulting in improved prognosis.

Citation Format: Mao Fujimoto, Eri Arai, Takuya Kikushima, Naotaka Nishiyama, Tomofumi Nakamura, Yuriko Yamada, Hiroaki Taira, Takuya Yotani, Hiroshi Kitamura, Yae Kanai. DNA methylation diagnostics for upper urinary tract urothelial carcinoma using newly developed high-performance liquid chromatography [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 6004.
Abstract 6009: An end-to-end workflow for improved methylation detection
Authors: Bonar Lydia , Butcher Kristin , Bocek Michael , Corbitt Holly , Hoglund Bryan , Nassif Cibelle , Cherry Patrick , Murphy Derek , Challacombe Jean , Toro Esteban
Published: April 2023

Introduction: DNA methylation at CpG nucleotide sites in eukaryotes is a key epigenetic mark that can help regulate gene expression. Specific changes in CpG methylation occur in many human cancers, making them a promising biomarker for early cancer detection. However, existing assays can be costly, lack specificity to regions of interest and often provide only semi-quantitative estimates of the methylation fraction. Here, we present a targeted methylome panel to decrease costs associated with next generation sequencing (NGS), methylation controls to calibrate quantitative assays and UMIs for accurate deduplication in low-diversity samples.

Experimental Procedures: Genomic DNA (gDNA) was prepared for sequencing using the Twist Methylation Detection System consisting of enzymatic methylation conversion and hybrid capture using the Twist Human Methylome Panel. Twist’s synthetic CpG methylation level controls were spiked-in to gDNA and taken through the Twist Methylation Detection System to demonstrate their utility in calibrating methylation assays. Additionally, libraries were generated using cell free DNA (cfDNA) and either conventional or UMI-containing adapters to investigate the impact on quantitative detection and total unique coverage.

Results: Using the Twist Human Methylome Panel at 150x raw coverage achieves uniform coverage with low off-bait Picard metrics. 6.59 million CpG sites were detected using a minimum depth of 10X. Target capture with the Human Methylome Panel allows for informative CpG calling of up to 82 samples on a single Illumina Novaseq S4 flowcell, compared to 3 samples per flowcell with a tradition whole-genome bisulfite sequencing (WGBS) assay. The Twist CpG methylation specific controls are constructed of 48 unique contrived sequences that contain a total of 8 different levels of methylation, ranging from 100% to 0%. Including these controls allows for quantitation of methylation levels in the experimental samples and qualification of the enzymatic conversion process.

Conclusions: Our study leverages the Twist methylation detection portfolio to interrogate genome-wide methylation patterns for various applications. In combination, these tools can be used to design cost effective end-to-end assays.

*For Research Use Only. Not for use in diagnostic procedures.

Citation Format: Lydia Bonar, Kristin Butcher, Michael Bocek, Holly Corbitt, Bryan Hoglund, Cibelle Nassif, Patrick Cherry, Derek Murphy, Jean Challacombe, Esteban Toro. An end-to-end workflow for improved methylation detection. [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 6009.
Abstract 6014: Cancer risk diagnosis by epimutation burden: a multicenter prospective study
Authors: Yamada Harumi , Maeda Masahiro , Abe Seiichiro , Shimazu Taichi , Ando Takayuki , Murakami Kazunari , Tanaka Shinji , Maekita Takao , Inatomi Osamu , Sugimoto Ken , Kawagoe Tetsuro , Yamamichi Nobutake , Ushijima Toshikazu
Published: April 2023

Aberrant DNA methylation, induced by aging and accelerated by chronic inflammation, accumulates in normal tissue. Accumulation of DNA methylation in multiple genes, namely epimutation burden, is closely associated with cancer risk. Indeed, we demonstrated that the measurement of the methylation level of a marker gene in normal tissue is useful for cancer risk prediction of metachronous cancers, by a multicenter prospective study with gastric cancer patients (Asada, Gut, 64:388, 2015: Maeda, Gut, 66:1721, 2017). In this study, we attempted to predict the risk of primary gastric cancer by performing a new multicenter prospective study. We recruited 1,880 healthy people, after Helicobacter pylori eradication, with a clinically high risk of gastric cancer. Biopsy specimens were obtained from the antrum and body, and the methylation level of RIMS1, which is a preselected epigenomic risk marker for this study, was measured in 1,757 participants. The participants are annually being followed-up to detect primary gastric cancer, for 5-8 years. Currently, the median follow-up is 4.05 years, and 27 people have developed primary gastric cancers (as of September 2022). The results of statistical analysis are being determined. This study will establish the clinical utility of a cancer risk diagnosis by epimutation burden in normal tissue, and is promising for application in various inflammation-associated cancers.

Citation Format: Harumi Yamada, Masahiro Maeda, Seiichiro Abe, Taichi Shimazu, Takayuki Ando, Kazunari Murakami, Shinji Tanaka, Takao Maekita, Osamu Inatomi, Ken Sugimoto, Tetsuro Kawagoe, Nobutake Yamamichi, Toshikazu Ushijima. Cancer risk diagnosis by epimutation burden: a multicenter prospective study. [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 6014.
Abstract 6073: Distinct subtype of multiple myeloma revealed by whole genome and transcriptome sequencing
Authors: Kim Sheehyun , Yoon Hyundong , Koh Youngil , Yoon Sung-Soo
Published: April 2023

Background: Advanced sequencing technologies have contributed to identifying genomic and transcriptomic features of various types of tumors. Despite several sequencing data from multiple myeloma (MM) being generated, understanding various properties of MM remained insufficient.

Methods: The patients confirmed with MM by bone marrow examination were analyzed in the study. Myeloma cells were enriched from the bone marrow aspirates of the patients using CD138. Whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) were performed for isolated myeloma cells. Mutational signatures were analyzed using somatic mutations detected by WGS.

Results: A total of 37 MM patients (median age 67) were enrolled in this study. WGS revealed somatic mutations of an average of 8872 single nucleotide variants (SNVs) and 934 insertions and deletions (Indels) from the patients, respectively. By the analysis of structural variants, 7 chromothripsis and 4 chromoplexy patients were identified. Mutational signature analysis showed that single base substitution (SBS) 9 signature largely varied between patients. SBS9 signature high patients were mostly diagnosed with non-IgG/non-IgA heavy chain and lambda light chain type myeloma. Notably, IgD myeloma patients were solely detected in the SBS9 signature high group compared to the low group (p = 0.001). Gene expression patterns obtained from WTS were well-divided into SBS9 signature high and low groups, and analysis of differentially expressed genes (DEG) between SBS9 signature high and low groups implied IgD myeloma features including high expression of IGHD. As a validation test, we tried mutational signature analysis on whole exome sequencing (WES) data of 784 MM patients from the MMRF database. Of the 38 patients with more than 500 exonic mutations, only the SBS9 signature high group had IgD myeloma candidates, whereas the SBS9 signature low group had no IgD myeloma candidate (p = 0.014).

Conclusions: DNA and RNA sequencing of myeloma patients could classify specific subtypes of disease categories. Further study for SBS 9 signature high and IgD myeloma is needed to discover the underlying mechanism of distinct features and to find out their clinical implication.

Citation Format: Sheehyun Kim, Hyundong Yoon, Youngil Koh, Sung-Soo Yoon. Distinct subtype of multiple myeloma revealed by whole genome and transcriptome sequencing. [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 6073.
Abstract 6097: Determination of BRCA2 deletion status by copy number estimation using next generation sequencing to predict prognosis of clinically localized prostate cancer
Authors: Sumitomo Makoto , Nukaya Takuhisa , Sugihara Eiji , Takeda Mayu , Nohara Sachio , Tanishima Shigeki , Takenaka Masashi , Zennami Kenji , Takahara Kiyoshi , Shiroki Ryoichi , Saya Hideyuki
Published: April 2023

The significance of BRCA alterations has been implicated in the development of metastatic castration-resistant prostate cancer (PC). On the other hand, the details of the frequency and significance of BRCA alterations in localized PC remain unknown. In this study, we investigated the frequency and clinical significance of BRCA alterations in localized PCs using an in-house next-generation sequencer (NGS) system. NGS analyses revealed that the BRCA2 CN decreased in 17 patients (13.5%) and the BRCA1 CN in six (4.8%). NGS-based CN values were shown to be highly correlated with droplet digital PCR-based CN values. Tissue-specific BRCA expression investigated using the Human Protein Atlas showed that the decreased CN of BRCA2, but not BRCA1, is responsible for the decreased BRCA activity in PC. Ten of the 22 patients with decreased BRCA2 CN were presumed to have somatic heterozygous deletion. There were no observed associations between the heterozygous deletion of BRCA2 and various clinicopathological parameters. Furthermore, three of 10 patients developed biochemical recurrence within 3 months after surgery. Multivariate analyses revealed that the initial prostate-specific antigen levels and BRCA2 CN were independent factors for biochemical recurrence. Our results suggest that a decrease in BRCA2 CN may be used as a biomarker for predicting recurrence after surgery in localized PC. Early screening for somatic alterations in BRCA2 using NGS may help to broadly predict the risk of PC progression.

Citation Format: Makoto Sumitomo, Takuhisa Nukaya, Eiji Sugihara, Mayu Takeda, Sachio Nohara, Shigeki Tanishima, Masashi Takenaka, Kenji Zennami, Kiyoshi Takahara, Ryoichi Shiroki, Hideyuki Saya. Determination of BRCA2 deletion status by copy number estimation using next generation sequencing to predict prognosis of clinically localized 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 6097.
Abstract 611: Sex disparities in cancer immunoediting
Authors: Hunt Katey , Alspach Elise
Published: April 2023

Cancer immunoediting is the process through which tumor specific T cell populations both prevent cancer development and apply selective pressure for the outgrowth of less immunogenic tumors with reduced neoantigen burden. Male and female immune systems demonstrate distinct differences that drive characterized disparities in response to viral infection and immunization. While several recent publications have shown that anti-tumor immune responses are inhibited by male sex hormone signaling, a critical gap in knowledge exists surrounding sex-based differences in the larger-scale process of immunoediting. Because the residual neoantigen burden expressed after tumor escape from immune surveillance is an important determinant of rejection following immune checkpoint blockade, disparities in immunoediting between males and females could have profound impacts on both cancer incidence and response to therapy. The purpose of our studies is to determine if the enhanced functionality of female T cells results in more effective immunoediting of tumors that evolve in females versus those that evolve in males. We have used the 3-methylcholanthrene carcinogen-induced tumor model and several transplant tumor models to determine if T cell function regulates differential cancer development between the sexes. We have also analyzed predicted neoantigen burden in male and female patients with melanoma and lung cancer to identify sex-based differences in tumor immunogenicity. We have found that female T cell responses are more effective at preventing tumor occurrence and delaying tumor outgrowth compared to male T cell responses in mice, which may help to explain established sex-based disparities in cancer incidence in the human population. In comparison, male patient tumors were more likely to express high neoantigen burden compared to their female counterparts. These results suggest that while stronger anti-tumor T cell responses in females are helpful in driving lower cancer incidence in females compared to males, this disparity may also result the development of tumors that express greater numbers of immunogenic neoantigens in males versus females. A better understanding of how sex impacts the entire process of immunoediting will help to explain cancer disparities between the sexes and identify potential new therapeutic strategies that can benefit all patients.

Citation Format: Katey Hunt, Elise Alspach. Sex disparities in cancer immunoediting [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 611.
Abstract 6151: Loss of GPR54 inhibits tumor growth by suppressing AKT/DDC signaling pathway in KRAS-mutant non-small cell lung cancer
Authors: Lee Seo Yeon , Choi Yu-Jeong , Hwang Hyun-Ha , Kang Sooyeon , Lee Kangwook , Ko Seong-Gyu
Published: April 2023

Therapeutic targeting of KRAS-mutant lung cancers has remained a challenge. There are increasing evidence that KISS1/GPR54 signaling pathway is associated with the development of various cancer. Although KISS1 and GPR54 are expressed in the lung, their biological role in the lung tumorigenesis remains unclear. In this study, using the genetically engineered mouse models, we have identified that loss of Gpr54 inhibited Kras-driven lung tumorigenesis by suppressing pathways in oncogenesis such as the MAPK and AKT pathways and inducing apoptosis. Similarly, genomic inhibition of GPR54 suppressed cell proliferation, decreased ERK and AKT signaling pathway, and increased apoptotic cell death in NSCLC cells. RNA-sequencing analysis revealed that DDC expression and glycolysis gene sets were significantly changed in Gpr54 deficient mice. Interestingly, HIF-1α expression is regulated by GPR54/AKT/DDC signaling pathway in NSCLC cells, leading to glycolytic change. Genomic inhibition of DDC suppressed cell proliferation and enhanced apoptotic cell death in NSCLC cell lines. Also, pharmacological inhibition of GPR54 and DDC lower glucose consumption and lactate production in NSCLC cell lines. Taken together, we demonstrate GPR54, as a potential therapeutic target, suggesting a new strategy to cure for lung cancer patients.

Citation Format: Seo Yeon Lee, Yu-Jeong Choi, Hyun-Ha Hwang, Sooyeon Kang, Kangwook Lee, Seong-Gyu Ko. Loss of GPR54 inhibits tumor growth by suppressing AKT/DDC signaling pathway in KRAS-mutant non-small cell lung 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 6151.
Abstract 6153: Triple negative breast cancer targeted by Ophiobolin A: Epithelial-mesenchymal transition take the lead
Authors: Ranganathan Santhalakshmi , Kornienko Alexander , Evidente Antonio , Romo Daniel , Taube Joseph
Published: April 2023

Triple negative breast cancer (TNBC) has fewer treatment options than other types of invasive breastcancer. Moreover, Epithelial to Mesenchymal Transition (EMT) helps enrichment of cancer stem cells(CSCs) which promotes resistance to chemotherapy. Ophiobolin A (OpA), a drug like small molecule wasrecently found to have anti-CSC properties. Our current study investigated the cell death mechanism(s)induced by OpA in various breast cancer cell lines with respect to epithelial-mesenchymal transition(EMT).The mechanism of cell death elicited by OpA was analyzed by applying inhibitors for specific pathwaysincluding apoptosis, necroptosis and paraptosis and by analyzing markers of cell death using flowcytometry and western blotting. HMLE(R) and HMLE(R)-Twist cell lines were used to assay the role of EMT.The formation of necrosomes was analyzed with exogeneous expression of RIPK3 tagged with greenfluorescence protein (GFP) in HeLa cell line which lacks endogenous RIPK3 expression. The necrosomeformation was identified by formation of puncta using confocal microscopy. Immune compromised SCIDmice were used to analyze the effect of OpA In vivo.The data obtained in the present study indicates that OpA has an increased anticancer effect in TNBCcompared to the estrogen receptor (ER) positive breast cancer cells. Cells having undergone EMT wereshown to have more sensitivity towards OpA when compared to -uninduced cell lines, which is sensitiveto Necrostatin-1, an inhibitor of necroptosis. OpA also diminished the CD44hi/CD24lo population in TNBCcells. However, apoptosis and paraptosis inhibitors failed to rescue cell death which clearly confirms thatOpA induces necroptosis cell death pathway in EMT undergone cells. An increased phosphorylation ofRIPK, a necroptosis protein marker, was observed on OpA treatment compared to the control. OpAtreatment significantly increased puncta TNBC compared to the untreated control cells, furtherconfirming the necroptotic cell death in TNBC. OpA treatment showed a significant tumor regression intumor bearing SCID mice. All together our findings identify that OpA induce necroptosis in EMT-enrichedTNBC. The study further needs an in-depth analysis of involvement of EMT in necroptotic cell death inTNBC.Key words: Triple-negative breast cancer, EMT, Ophiobolin A, Necroptosis, cell deathmechanism

Citation Format: Santhalakshmi Ranganathan, Alexander Kornienko, Antonio Evidente, Daniel Romo, Joseph Taube. Triple negative breast cancer targeted by Ophiobolin A: Epithelial-mesenchymal transition take the lead. [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 6153.
Abstract 6388: High dose acetaminophen with n-acetylcysteine rescue inhibits M2 polarization of tumor associated macrophages
Authors: Neuwelt Alexander , Bryan Allyn , Lee Won , Pingali Pavani , Li Howard , Patel Bhaumik
Published: April 2023

Background: High dose acetaminophen (AAP) with n-acetylcysteine (NAC) rescue is among the few treatments that has shown a signal of activity in phase I trials (21% response rate, 3/14 assessable patients) without achieving dose limiting toxicity that has not progressed to evaluation in later line studies. We have previously shown that high dose AAP selectively depletes glutathione in the liver but not the tumor, suggesting that NAC, the FDA-approved antidote for AAP toxicity and a glutathione precursor, may selectively rescue the normal liver but not the tumor from high dose AAP. While the anti-tumor effects of AAP/NAC appear to not be mediated by glutathione depletion and free radical injury, the mechanism of anti-tumor effects of AAP/NAC has not been definitively characterized.

Methods: In vitro the effects of Vehicle, AAP, or AAP/NAC was evaluated on bone marrow derived macrophages polarized to M2 and M1 phenotypes using IL-4 or IFN/LPS, respectively. Tumor cell viability was assessed with MTT assay. Effects of AAP on IL-4/STAT6 (M2) or IFN/LPS/STAT1 (M1) signaling, and downstream gene and protein expression was studied. In vivo, effects of AAP/NAC on tumor size and tumor associated macrophage polarization were characterized in orthotopic triple negative breast tumor models 4T1 and EF43.fgf4 using cytokine elisa of tumor lysate and flow cytometry.

Results: NAC reversed AAP toxicity in the normal liver but did not reverse AAP cytotoxicity against tumor cells in vitro. AAP/NAC selectively inhibited IL-4-induced STAT6 phosphorylation but not IFN/LPS-induced STAT1 phosphorylation. Downstream, AAP/NAC inhibited IL-4 induction of M2-associated genes (such as CCL24, YM1 and arginase) and proteins (arginase, PD-L2, and CD206). However, AAP/NAC did not inhibit IFN/LPS induction of M1-associated genes (IL-6, TNF, IL-12) and proteins (INOS, PD-L1, MHC I, and CD64). In vivo, AAP/NAC inhibited tumor growth in EF43.fgf4 and 4T1 triple negative breast tumors. Flow cytometry of tumor associated macrophages revealed that AAP/NAC selectively inhibited M2 but not M1 polarization. ELISA of tumor lysate demonstrated relatively stable expression of IFN gamma but markedly suppressed expression of IL-10 in the tumor immune microenvironment.

Conclusion: Our study is the first to show that at high doses AAP/NAC has profound effects on the tumor immune microenvironment that may facilitate immune-mediated inhibition of tumor growth.

Citation Format: Alexander Neuwelt, Allyn Bryan, Won Lee, Pavani Pingali, Howard Li, Bhaumik Patel. High dose acetaminophen with n-acetylcysteine rescue inhibits M2 polarization of tumor associated macrophages [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 6388.
Abstract 6424: Immune characteristics and anti-PD-1-induced reinvigoration capacity of tumor-infiltrating lymphocytes in ovarian clear cell carcinoma relative to high-grade serous ovarian carcinoma
Authors: Park Junsik , Lee Yong Jae , Kim Jung Chul , Lee Jung-Yun
Published: April 2023

Recent clinical trials have demonstrated the potential efficacy of immune checkpoint inhibitors (ICIs) for ovarian clear cell carcinoma (OCCC). However, little is known about the immune characteristics of OCCC. In this study, we aimed to investigate the distinct immune characteristics of tumor-infiltrating lymphocytes (TILs) in OCCC to high-grade serous ovarian carcinoma (HGSOC). We isolated peripheral blood mononuclear cells (PBMCs) and TILs from patients with epithelial ovarian cancer. The expression of immune checkpoint receptors and T-cell transcription factors of TILs in OCCC (n=26) and HGSOC patients (n=44) were accessed using flow cytometry. TILs were ex vivo stimulated with anti-CD3 in the presence of anti-PD-1 and/or anti-CTLA-4, and their proliferation was assessed to predict response to ICIs. CD8 TILs and tumor-infiltrating regulatory T cells (Tregs) exhibited higher expression of immune checkpoint receptors, including PD-1, CTLA-4, and 4-1BB, compared to PBMCs. Tregs were more infiltrated in advanced/recurrent stage than early stage OCCC. However, immune characteristics of CD8 TILs and Tregs were not significantly different regardless of stage. When we compared the characteristics of TILs in OCCC (n=26) with those in HGSOC (n=44), the expression of PD-1 on CD8 TILs was significantly lower in OCCC than in HGSOC. The frequencies of PD-1highCD8 TILs and PD-1+TOX+CD8 TILs were also lower in OCCC than HGSOC. In ex vivo assay (n=19), 9 cases responded to anti-PD-1 in terms of enhanced proliferation capacity of CD8 TILs. In addition, when anti-CTLA-4 was added to anti-PD-1, an additional increase in proliferation of CD8 TILs was observed in patients who responded to anti-PD-1. Responders to anti-PD-1 had significantly lower frequencies of effector Tregs in the TILs than non-responders. Overall, CD8 TIL in OCCC was found to be less exhausted than HGSOC in terms of PD-1 and TOX expressions. Tumor-infiltrating Tregs affected on reinvigoration ability of CD8 TILs upon ICIs treatment in OCCC. This study provides the rationale and evidence for establishing the optimal strategies for ICIs treatment in patients with OCCC.

Citation Format: Junsik Park, Yong Jae Lee, Jung Chul Kim, Jung-Yun Lee. Immune characteristics and anti-PD-1-induced reinvigoration capacity of tumor-infiltrating lymphocytes in ovarian clear cell carcinoma relative to high-grade serous ovarian 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 6424.
Abstract 6529: Early life liver proteomic signature and minerals mitigation of liver injury
Authors: Harber Isabelle , McClintock Shannon D. , Knibbs Randall N. , Varani James , Aslam Muhammad N.
Published: April 2023

Background and Aims: Previous long-term murine (12- and 18-month) studies in C57BL/6 mice have demonstrated the beneficial role of Aquamin, a red marine algae-derived multi-mineral supplement containing calcium, magnesium, and 72 additional trace elements, in inhibiting liver tumor formation in mice on high-fat, Western-style diet (HFWD). Liver tumor incidence in mice on HFWD was decreased to 12% with the addition of Aquamin supplementation compared with 48% in the HFWD mice. Similarly, in mice on the low-fat diet, tumor incidence was only 16% and Aquamin further reduced it to 4%. Recently, we assessed the safety and tolerability of Aquamin in human subjects in a 90-day interventional trial and there was no evidence of potential liver harm, measured by liver enzymes. A short-term (16-week) mouse study was carried out to investigate early protein changes in the liver that may play a role in liver tumorigenesis and how Aquamin can interfere with that signature.

Methods: Male MS-NASH mice were maintained on a HFWD for 16 weeks with and without red algae-derived minerals. A cohort of C57BL/6 mice maintained on a standard chow (low-fat diet) was used as a control. At the end of the in-life portion of the study, body weight, liver weight, liver enzyme levels and liver histology were assessed. Samples obtained from individual livers were subjected to Tandem Mass Tag labeling and quantitative proteomics mass spectrometry.

Results: As expected, no tumors were observed at 16 weeks. Mice on the HFWD demonstrated multiple protein changes associated with dysregulated fat and carbohydrate metabolism, lipotoxicity and oxidative stress. In mice receiving Aquamin-supplementation along with the HFWD, there was reduced liver toxicity as evidenced by a decrease in levels of several cytochrome P450 enzymes and other oxidant-generating moieties. Additionally, multiple keratins, which provide cytoskeletal support were elevated. Phosphate and tensin homolog (PTEN), a tumor suppressor, was upregulated with Aquamin. There was an upregulation of several pathways implicated in tumor suppressor activity, notably keratinization, hedgehog signaling in the off-state, and regulation of PTEN stability and activity.

Conclusions: The proteomic expression of the livers of HFWD mice in this short-term study explains early protein changes that may contribute to liver cancer and the potential mechanisms by which dietary minerals may mitigate such down-stream effects.

Citation Format: Isabelle Harber, Shannon D. McClintock, Randall N. Knibbs, James Varani, Muhammad N. Aslam. Early life liver proteomic signature and minerals mitigation of liver injury [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 6529.
Abstract 6578: reVUE: repository for variants with unexpected effects
Authors: de Bruijn Ino , Gopalan Aditi , Li Xiang , Zhang Hongxin , Sumer Onur , Cong Thomas Y. , Gonzalez Tona , Rangel Madelaine , Schultz Nikolaus , Chakravarty Debyani
Published: April 2023

Clinical sequencing of tumor samples is now a component of routine cancer care used to identify predictive biomarkers of drug response, refine patient cancer diagnoses, assess heritable cancer risk, or inform patient prognosis. Most genomic alterations are accurately annotated with tools such as the Variant Effect Predictor (VEP) that infer the effects of these alterations on the mRNA and protein by following basic rules of transcription, mRNA post-transcriptional processing, and translation. However, some genomic variants are not as easily captured by these rules, which can cause inappropriate or unclear annotation of the protein effect. For example, a variant that alters an existing splice site or creates a new one can activate or inactivate the protein. Similarly, specific mutations in the non-coding promoter region of a gene may de-regulate gene expression (e.g., TERT promoter mutations). In some instances, these so-called variants with unexpected effects (VUE) may have therapeutic implications. For example, while ~2-3% of GIST tumors harbor KIT exon 11 deletions that extend into the non-coding intron between exons 10 and 11 and are shown to cause in-frame deletions, they are typically misclassified as inactivating splice site mutations, precluding patients with tumors harboring these mutations from receiving standard care imatinib.

Thus, while many VUEs are functionally characterized and documented in the literature, there are no resources to systematically identify, curate and store these events such that they can be correctly annotated in routine clinical sequencing leading to suboptimal treatment decisions.

To address this unmet clinical need, we built a novel bioinformatic resource, the Repository for Variants with Unexpected Effects (reVUE - cancerrevue.org). The reVUE resource consists of (1) an intuitive website listing curated VUEs with their observed effects as demonstrated by functional characterization in peer-reviewed literature and (2) an application programming interface (API) for programmatic annotation of variants. We built reVUE as a crowd-sourced resource to enable contributions of VUEs to the repository from the clinical and scientific communities. Both the software and data are openly available. Our goal is to enable clinical bioinformatic pipelines to accurately annotate all DNA mutations, including those with unanticipated protein coding effects, some of which can have therapeutic implications.

Citation Format: Ino de Bruijn, Aditi Gopalan, Xiang Li, Hongxin Zhang, Onur Sumer, Thomas Y. Cong, Tona Gonzalez, Madelaine Rangel, Nikolaus Schultz, Debyani Chakravarty. reVUE: repository for variants with unexpected effects. [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 6578.
Abstract 6592: An AI platform for vascular and fibrosis analysis of pathology images
Authors: Zheng Yawen , Zhang Likun , Wang Dawei , Gao Sheng
Published: April 2023

Introduction: Changes in the number of blood vessels and the proportion of fibrosis regions in xenograft tumors are key indicators for efficacy of oncology drugs, which currently are assessed mainly by human visual examination that is time-consuming, inaccurate and biased because only part of a pathology image is checked. We developed an online AI platform that integrates machine learning algorithms in Computer Vision to quickly and automatically obtain accurate and comprehensive information on blood vessels and fibrosis regions from a whole pathology image.

Methods: In a stained pathology image, blood vessels are stained with a different color than other components (e. g. connective tissues, tumor cells, background), so we can directly locate pixels representing them. They also have different topological structures, so we can outline and count the contours of vessels. To count the number of vessels more precisely and reduce the interference of noise, we keep a contour only if it intersects with some located pixels and the number of pixels in the intersection exceeds an adjustable threshold. The fibrosis regions are identified by color contrast since they are stained with a distinct and easily recognizable color. The algorithms were implemented using the OpenCV package.

Results: Our AI platform automatically annotates blood vessels and fibrosis regions in pathology images, and generates reports in excel format to report the number and total area of blood vessels, the area and ratio of fibrosis regions, and execution time. It demonstrates superior performance to standard pathology software.

Citation Format: Yawen Zheng, Likun Zhang, Dawei Wang, Sheng Gao. An AI platform for vascular and fibrosis analysis of pathology images [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 6592.