bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5895: Analysis of microbiome in gastric MALT lymphoma
Authors: Yoo SeungJoo , Shin Dong-Yeop , Byun Ja Min , Hong Junshik , Yun Hongseok , Koh Youngil , Yoon Sung-Soo
Published: April 2023

Gastric MALT (mucosa-associated lymphoid tissue) lymphoma is found at a rate of 50% or more among the types of MALT lymphoma. Majority of patients with gastric MALT lymphoma are infected with Helicobacter pylori and also have clinical improvement with eradication therapy. However, it is unclear how H. pylori cause gastric MALT lymphoma. To explore it, we prospectively collected 13 patients who are newly diagnosed with gastric MALT lymphoma with H. pylori. We gathered samples from normal regions and lesions during endoscopy for diagnosis. Samples of lesion were harvested again after eradication of H. pylori. we sequenced 10 of WES (whole exome sequencing) and 13 of WTS (whole transcriptome sequencing) with these samples. With sequencing data, we aligned the sequenced reads to human reference genome (hg19) using BWA and called the variants with Mutect2. We aligned the reads and calculated TPM (Transcripts Per Million) from WTS with STAR and RSEM. We performed DEG (Differentially Expressed Genes) and GSEA (Gene Set Enrichment Analysis) for three comparison groups which are divided into normal, lesion, and after eradication. Gene fusions were detected using STAR-Fusion and Arriba with WTS. To detect potential microbiome from the samples, we used Kraken2, a taxonomic classification tool. We found that MUC6 was mutated from all samples and some samples have mutated MUC3A, KMT2C, ZNF595, CDC27, etc., and two have BIRC3-MALT1 fusion. From DEG and GSEA analysis, G protein-coupled receptor activity was activated in the group of lesion samples and suppressed in normal samples and eradication samples. Lymphocyte activation and immune response were also activated in the cancerous lesion. Kraken2 classified the reads which were not aligned to the human reference genome as reads from microbiota including H. pylori. The reads of H. pylori were more abundant in the lesion group than the other groups. According to the result of differentially expressed species analysis between the groups of lesion and eradication, the following species were also more abundant in the lesion group; Veillonella dispar, Helicobacter hepaticus, Helicobacter winghamensis, etc. We analyzed genetic alterations and differentially expressed genes and also classified non-human reads as microbiome with sequencing data from gastric MALT lymphoma. Our results suggest how H.pylori infection is related to carcinogenesis of gastric MALT lymphoma.

Citation Format: SeungJoo Yoo, Dong-Yeop Shin, Ja Min Byun, Junshik Hong, Hongseok Yun, Youngil Koh, Sung-Soo Yoon. Analysis of microbiome in gastric MALT lymphoma [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 5895.
Abstract 5896: Identification of microbial biomarkers to predict recurrence of oral squamous cell carcinoma
Authors: Lyu Wei-Ni , Lin Mei-Chun , Lou Pei-Jen , Lai Liang-Chuan , Tsai Mong-Hsun
Published: April 2023

Introduction: Oral cancer is a fatal cancer and the sixth most common neoplasm worldwide. Over 90% of oral cancer is oral squamous cell carcinoma (OSCC). OSCC is a global health problem because of its late diagnosis and high rate of relapse and metastasis. Currently, cumulative evidence has suggested the association between oral microbiome and oral cancer. However, the correlation between the oral microbiome and OSCC recurrence remained unclear. Therefore, we investigated the OSCC patients’ oral microbiota to understand the roles of oral microbiome in the recurrence of OSCC.

Materials and Methods: To evaluate the role of the oral microbiome in the recurrence of oral cancer, we compared the oral bacterial composition of tumor samples from the OSCC patients with or without recurrence based on 16S rRNA amplicon sequencing of 54 oral swab samples. Then, the structures of the oral microbiome were analyzed to establish a prediction model for the recurrence of OSCC. To identify microbial signatures capable of distinguishing recurrence from non-recurrence, the 54 tumor samples from OSCC patients were used as a training dataset. Furthermore, 46 external tumor samples from other OSCC patients were used to validate the dataset.

Results: The oral bacterial compositions differed in OSCC patients between with and without recurrence. Compared to nonrecurrence OSCC, periodontitis-related bacteria were enriched in OSCC recurrence. Functional analysis of the oral microbiome showed several functions associated with OSCC recurrence including amino acid metabolism, carbohydrate metabolism, lipid metabolism, glucose utilization, and drug resistance. Subsequently, we established a random forest prediction model with high accuracy (accuracy = 0.963) to discriminate OSCC recurrence from the original OSCC based on five specific microbial signatures. Moreover, the prediction model achieved an accuracy of 0.761 in another independent cohort (46 OSCC patients). On the other hand, the accuracy predicted by the current clinical-used model was 0.519 with the training dataset. Thus, our novel model could improve the prediction accuracy of OSCC recurrence.

Conclusions: In this study, we elucidated the relationship between oral bacteria and the recurrence of OSCC. Moreover, we established a novel bacteria-based prediction model for the recurrence of OSCC. Thus, the present study provided a new insight and treatment strategy for the clinical prediction of OSCC recurrence.

Citation Format: Wei-Ni Lyu, Mei-Chun Lin, Pei-Jen Lou, Liang-Chuan Lai, Mong-Hsun Tsai. Identification of microbial biomarkers to predict recurrence of oral squamous cell 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 5896.
Abstract 5998: Global DNA methylation analysis of cancer-associated fibroblasts reveals extensive epigenetic rewiring linked with RUNX1 upregulation in breast cancer stroma
Authors: Halperin Coral , Hey Joschka , Weichenhan Dieter , Stein Yaniv , Mayer Shimrit , Lustik Pavlo , Plass Christoph , Scherz-Shouval Ruth
Published: April 2023

Cancer cells recruit and rewire normal cells in their microenvironment to support and protect them by creating a pro-tumorigenic tumor microenvironment (TME). We lack an overarching view of how, despite being genomically stable, stromal cells in the tumor microenvironment are heterogeneously reprogrammed across time and space to promote the evolution of aggressive disease. Recent work by us and others has shown that fibroblasts in the tumor microenvironment are transcriptionally rewired to become protumorigenic cancer associated fibroblasts (CAFs). Transcriptional regulation plays a major role in this reprogramming, but the extent to which epigenetic modifications of DNA also contribute to the rewiring of CAF transcription is not clear. Here we address this question by dissecting the epigenetic landscape of breast CAFs. We applied a sensitive method of whole genome bisulfide sequencing on a model of triple-negative breast cancer in mice to evaluate the methylome profile of CAFs compared to normal mammary fibroblasts (NMFs). we found that fibroblasts undergo massive DNA methylation changes as they transition into CAFs in mice. These changes inversely correlated with transcriptional changes between CAFs and NMFs. We characterized potential regulators of this process, and tested their expression in CAFs in human breast cancer patients, to confirm relevance of our findings to human disease. Our findings suggest that epigenetic alterations contribute to the transcriptional rewiring of fibroblasts to CAFs. This work presents a comprehensive map of DNA-methylation in CAFs, and reveals a previously unknown facet of the dynamic plasticity of the stroma.

Citation Format: Coral Halperin, Joschka Hey, Dieter Weichenhan, Yaniv Stein, Shimrit Mayer, Pavlo Lustik, Christoph Plass, Ruth Scherz-Shouval. Global DNA methylation analysis of cancer-associated fibroblasts reveals extensive epigenetic rewiring linked with RUNX1 upregulation in breast cancer stroma [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 5998.
Abstract 6005: Promoter methylation occurs at high frequencies regardless of mutation status of tumor-associated genes in oral malignancies
Authors: Abe Masanobu , Hoshi Kazuto , Ushijima Toshikazu
Published: April 2023

Background: Genetic and epigenetic alterations are both important in tumorigenesis. However, their association in oral carcinogenesis has not been fully investigated yet. In this study, we analyzed somatic mutations of tumor-related genes and promoter methylation of tumor-suppressor genes (TSGs) in oral malignancies/premalignancies, and analyzed their association.

Materials and Methods: 39 oral squamous cell carcinomas (OSCCs) and 2 oral epithelial dysplasias (OEDs) were obtained as fresh-frozen?. Somatic mutations of 46 tumor-related genes, 13 TSGs and 33 oncogenes, were analyzed by targeted next-generation sequencing (NGS). Promoter methylation of five TSG: (p16/INK4A, CMTM3, EGFLAM, NKX2-3, and RBP4) were determined by methylation-specific PCR (MSP).

Results: Somatic mutations of tumor-related genes were observed in 63.4% of 41 OSCCs/OEDs. Particularly, mutations of p53 and PIK3CA were observed 39.0% and 31.7%, respectively, of the 41 OSCCs/OEDs. Promoter methylation of p16/INK4A, CMTM3, NKX2-3, RBP4, and EGFLAM were observed in 34.1%, 48.8%, 41.5%, 68.3%, and 36.6%, respectively, of the 41 OSCCs/OEDs. No differences in the frequency of promoter methylation of the five genes were observed between tumors with and without somatic mutations of p53 and PIK3CA.

Conclusions: Promoter methylation was observed at high frequencies regardless of mutation status of p53 and PIK3CA in OSCCs/OEDs. Oral tumorigenesis appears to be difficult to be explained solely by genetic events indicating involvement of epigenetic events.

Citation Format: Masanobu Abe, Kazuto Hoshi, Toshikazu Ushijima. Promoter methylation occurs at high frequencies regardless of mutation status of tumor-associated genes in oral malignancies [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 6005.
Abstract 6007: p16 epimutation modulates the tumor microenvironment and promotes malignancy of Apc-mutant colon cancer
Authors: Yang Li , Chen Xiaomin , Lee Christy , Shi Jiejun , Lawrence Emily B. , Zhang Lanjing , Li Yumei , Gao Nan , Jung Sung Yun , Creighton Chad J. , Li Jingyi Jessica , Cui Ya , Arimura Sumimasa , Li Wei , Shen Lanlan
Published: April 2023

Methylation of the p16 promoter resulting in mitotically stable gene silencing—known as p16 epimutation—is the most common epigenetic event in human colorectal cancer (CRC) and is also common in normal-appearing colonic mucosa of aging individuals. However, the functional role of p16 epimutation in CRC carcinogenesis is not completely understood. Here, using a mouse model of engineered p16 promoter hypermethylation, we show that p16 epimutation cooperates with mutant Apc to promote colon cancer development. Mice carrying combined Apc mutation and p16 epimutation display significantly shortened survival and increased tumor growth compared to those with Apc mutation only. Moreover, colon tumors from these animals express increased levels of programmed death-ligand 1 (Pdl1). Single-cell RNA sequencing (scRNA-seq) analysis further revealed the presence of dysfunctional cytotoxic CD8+ T cells together with Foxp3+ Tregs and γδT17 cells, which promote an immunosuppressive tumor microenvironment (TME). Lastly, we show that a combined therapy using an inhibitor of DNA methylation and a PD-L1 immune checkpoint inhibitor is more effective for improving survival in tumor-bearing mice than anti-PD-L1 treatment alone. These findings provide a mechanistic rationale for targeted epigenetic and immunotherapy in CRC patients, with the potential to improve colon cancer treatment.

Citation Format: Li Yang, Xiaomin Chen, Christy Lee, Jiejun Shi, Emily B. Lawrence, Lanjing Zhang, Yumei Li, Nan Gao, Sung Yun Jung, Chad J. Creighton, Jingyi Jessica Li, Ya Cui, Sumimasa Arimura, Wei Li, Lanlan Shen. p16 epimutation modulates the tumor microenvironment and promotes malignancy of Apc-mutant colon 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 6007.
Abstract 6015: Methylation synthetic lethality: CHFR methylation and KRAS inhibition in gastric cancers
Authors: Takeshima Hideyuki , Ebata Takahiro , Furuichi Yumi , Yamashita Satoshi , Ushijima Toshikazu
Published: April 2023

Synthetic lethality is a promising therapeutic strategy, but the number of founder mutations is limited. In contrast, DNA methylation is frequently present in many types of cancers, and can be a potential target for a synthetic lethality. So far, BRCA1 methylation with a PARP inhibitor is known as a synthetic lethality [Kawachi et al., Breast Cancer Res Treat, 181:323-329, 2020]. In this study, we aimed to identify a novel methylation synthetic lethal combination in gastric cancers. Founder methylation was searched for in 8,008 genes with putative promoter CpG islands (CGIs), and 137 genes which were 1) heavily methylated and 2) frequently methylated in primary gastric cancers (10 or more of 50 cases) were selected. The association analysis between DNA methylation and gene expression showed that 10 of 137 genes were completely silenced in gastric cancer cell lines with methylation of their promoter CGIs, but were expressed in those without. Among these 10 genes, we focused on CHFR, a known tumor-suppressor gene in gastrointestinal cancers, and genes lethal for CHFR methylation were screened for using the sgRNA screening database (The Cancer Dependency Map, DepMap). Comparison of gene dependencies of 17,645 genes between four cell lines with CHFR methylation and 12 cell lines without revealed that knockout of KRAS reduced cell growth specifically in cell lines with methylation. This synthetic lethality could be experimentally confirmed in two cell lines with CHFR methylation, AGS and 44As3, and three cell lines without, HSC41, HSC42, and MKN74. These cell lines included those not used in the screening, validating the usefulness of the combination. These results showed that gastric cancers with CHFR methylation were sensitive to KRAS inhibition, and that methylation synthetic lethality is a promising therapeutic strategy.

Citation Format: Hideyuki Takeshima, Takahiro Ebata, Yumi Furuichi, Satoshi Yamashita, Toshikazu Ushijima. Methylation synthetic lethality: CHFR methylation and KRAS inhibition in gastric 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 6015.
Abstract 602: Single cell evaluation of intratumoral B cells across solid tumor indications
Authors: Fahl Shawn P. , Colwell Kerri , Smith Casey , Ott Cavin , Moore Alex , Williams Kayla
Published: April 2023

The immune system is a critical component of the tumor microenvironment that has both tumor-promoting and tumor-eradicating activity based on the specific immune cell subtype and their activation status. T lymphocytes represent the largest lymphocyte population within the tumor, and numerous immunotherapies have been developed to specifically target this cellular subset. B cells are the next most abundant lymphocyte subset within the tumor microenvironment, and recent studies have identified crucial roles for intratumoral B cells in the production of tumor-reactive antibodies. Using human dissociated tumor cells (DTCs) samples, we have profiled intratumoral B cells to determine their frequency and subset breakdown across thirteen different solid tumor indications. Intratumoral B cells were prevalent in lung cancer and cancers of the gastrointestinal tract (esophageal, gastric, and colorectal), while they were rarer in cancers of the female reproductive tract (endometrial and ovarian). The transcriptional profile and B cell repertoire of the intratumoral B cell subsets were evaluated to provide a deeper understanding of the biology of these tumor-infiltrating lymphocyte subsets. Additionally, spatial transcriptomics were utilized to understand the spatiotemporal location of these subsets within the tumor microenvironment. These studies will provide critical insight for the development of the next generation of immunotherapies.

Citation Format: Shawn P. Fahl, Kerri Colwell, Casey Smith, Cavin Ott, Alex Moore, Kayla Williams. Single cell evaluation of intratumoral B cells across solid tumor indications [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 602.
Abstract 6040: The CHST11-CP-iron axis contributes to NSCLC stemness
Authors: Li Li-Jie , Chang Wei-Min , Hsiao Michael
Published: April 2023

The emerging advances in cancer therapies have changed the landscape for the treatment, but there are limited treatments options for advanced non-small-cell lung cancer (NSCLC). Cancer stem cell-induced chemoresistance and tumor metastasis accounts for treatment failure and recurrence in NSCLC. Additionally, the up-regulated glycosaminoglycan-chondroitin sulfate (CS) biosynthesis pathway contributes to the mesenchymal state of lung cancer and related to unfavorable prognosis. Previously, we found carbohydrate sulfotransferase 11 (CHST11), a key CS biosynthetic enzyme, was associated with tumor progression and metastasis in NSCLC as well as lung fibrosis. Furthermore, ceruloplasmin (CP) acted as a downstream effector of CHST11 through interferon-γ signaling pathway stimulation and increased NSCLC metastatic features. However, the linkage between metabolic dysregulation and NSCLC stemness remains unclear. Here, we found not only overexpression of CHST11 but also CS-A treatment, the metabolite of CHST11, promoted NSCLC tumor sphere formation, the expression of cancer stemness markers, and transcriptional activities of stemness-related pathways. Iron was a necessary ion in upregulating cancer stemness properties such as tumor-initiation, metastasis and chemoresistance. We found both CHST11 and iron enhanced NSCLC tumorigenesis. Moreover, iron chelation disrupted CHST11-CP axis induced-stemness. The combination of iron chelator with chemotherapies showed the synergistic effect on antitumor as well as anti-cancer stemness and further promoted apoptosis. Our findings implicate that the CHST11-CP-iron axis facilitates tumor progression and cancer stemness in NSCLC. A regimen that combines an iron chelator and chemotherapies targeting cancer stem cells will benefit for not only preventing NSCLC tumorigenesis but cancer recurrence.

Citation Format: Li-Jie Li, Wei-Min Chang, Michael Hsiao. The CHST11-CP-iron axis contributes to NSCLC stemness. [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 6040.
Abstract 6047: ZFTA-RELA fusion aberrantly drives glutamine metabolism in lethal pediatric ependymomas
Authors: Natarajan Siva Kumar , Haggerty-Skeans James , Lum Joanna , Narayanan Pranav , Sweha Stefan , Sunil Sushanth , Panwalkar Pooja , Bayliss Jill , Sajjakulnukit Peter , Dang Derek , Achreja Abhinav , Nagrath Deepak , Lyssiotis Costas , Venneti Sriram
Published: April 2023

Ependymomas are fatal brain malignancies with very few treatment options. More than 70% of supratentorial (ST) ependymomas harbor fusions of the zinc-finger containing, chromatin modifier ZFTA and the transcriptional activator of NF-κB signaling, RELA. Oncogene-driven metabolic reprogramming is a fundamental hallmark of cancer that enables sustained tumor proliferation. ZFTA-RELA fusion protein is essential for tumorigenesis and our goal is to determine how it drives metabolism in ST-ependymomas. To address this, we developed an in vitro isogenic system by expressing the ZFTA-RELA fusion protein in immortalized mouse neural stem cells (ZFTA-RELAFUS). Using this system, we show that these tumor cells selectively upregulate expression of the glutamine transporter (SLC1A5), glutaminase (GLS), and many downstream enzymes in the glutamine metabolic pathway. Therefore, we hypothesized that the ZFTA-RELA fusion drives glutamine metabolism in ST-ependymomas. We further demonstrate that ZFTA-RELAFUS tumor cells utilize glutamine to maintain redox homeostasis and show marked cell death upon its withdrawal. Moreover, JHU-083, a specific pharmacologic inhibitor of glutaminase, killed ZFTA-RELAFUS tumor cells in vitro and in vivo. To summarize, our results suggest that the ZFTA-RELA fusion expressing tumor cells exhibit strong glutamine dependence, and targeting it has significant therapeutic relevance.

Citation Format: Siva Kumar Natarajan, James Haggerty-Skeans, Joanna Lum, Pranav Narayanan, Stefan Sweha, Sushanth Sunil, Pooja Panwalkar, Jill Bayliss, Peter Sajjakulnukit, Derek Dang, Abhinav Achreja, Deepak Nagrath, Costas Lyssiotis, Sriram Venneti. ZFTA-RELA fusion aberrantly drives glutamine metabolism in lethal pediatric ependymomas [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 6047.
Abstract 6070: Comprehensive real-world-data based molecular profiling and mapping of non-squamous NSCLC patients to immune-checkpoint-inhibitor biomarkers
Authors: Kloetgen Andreas , Wang Danyi , Coenen-Stass Anna , Gounaris Ioannis , Hopkins Julia F. , Locatelli Giuseppe , Scheuenpflug Juergen , Feng Zheng
Published: April 2023

Purpose: Non-squamous non-small cell lung cancer (nsqNSCLC) patients without detectable actionable alterations in EGFR, ALK, or ROS1, have a diverse genetic background. These patients are eligible to and usually receive untargeted, first line immune checkpoint inhibitors (ICI) and/or chemotherapy with heterogeneous outcomes. To identify potential predictive biomarkers for the stratification of nsqNSCLC patients without actionable genetic alterations, a comprehensive molecular profiling of this subpopulation was performed using the Foundation Medicine genomic database.

Methods: Molecular profiles from 53,119 nsqNSCLC patients were analyzed using the FoundationInsightsTM web platform. After the patients with known and likely functional short variants for EGFR and fusions for ALK and ROS1 were excluded, the prevalences for commonly altered genes were determined. Pathway analysis was conducted with Metascape. Statistical significance and enrichment in PD-L1 and tumor mutation burden (TMB) biomarker overlap with altered genes was calculated using a fisher exact test against an alteration negative cohort.

Results: nsqNSCLC patients without actionable genetic alterations can be described as a heterogenous population with different genetic alterations. We found a total of 103 genes with an individual, minimal prevalence of >1% to be the major descriptors that collectively were found in most patients of the nsqNSCLC subpopulation. These genes relate to pathways involved in cell cycle, cell proliferation, response to growth factors or chromatin organization. Out of the most frequently altered genes, we identified those that are associated with PD-L1 or TMB high status for which ICI treatment is most effective in nsqNSCLC.

Conclusions: The results supported the lack of a general association with high PD-L1 and high TMB calls for biomarker informed treatment options for nsqNSCLC patients. We reported a subpopulation of nsqNSCLC patients that can be described by certain genetic alteration, of which some were associated with ICI-related biomarkers for which patients might benefit from a future precision oncology approach utilizing combined targeted and ICI treatment.

Citation Format: Andreas Kloetgen, Danyi Wang, Anna Coenen-Stass, Ioannis Gounaris, Julia F. Hopkins, Giuseppe Locatelli, Juergen Scheuenpflug, Zheng Feng. Comprehensive real-world-data based molecular profiling and mapping of non-squamous NSCLC patients to immune-checkpoint-inhibitor biomarkers. [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 6070.
Abstract 6107: Novel roles for AMBRA1 in regulating DNA double-strand breaks
Authors: Jiang Yuning , Abbas Tarek
Published: April 2023

AMBRA1 (Activating Molecule in Beclin-1-Regulated Autophagy) is a tumor suppressor protein whose expression is downregulated in several human malignancies. AMBRA1 was recently found to function as a substrate receptor for the cullin-4-based ubiquitin ligase (CRL4AMBRA1) that promotes the ubiquitin-dependent proteolysis of D-type cyclins (cyclin D1, D2 and D3); this serves to guard against premature S-phase entry and replication stress, providing a potential mechanism for its tumor suppressive activity. Cyclin D1 protein, which is stabilized, along with cyclin D2 and D3, in AMBRA1-deficient cells, has been shown to promote homologous recombination (HR) repair of DNA double-strand breaks (DSBs), suggesting that AMBRA1 may be additionally involved in the repair of DSBs. Using our recently published CRISPR-Cas9-based dual fluorescent reporter assay system, we show that while the loss of AMBRA1 increased D-type cyclins and Rb hyperphosphorylation as expected, it did, surprisingly, inhibit HR-mediated repair and stimulated error-free non-homologous end-joining (NHEJ), and that both of these new activities are independent of cyclin D1 or RB1. High-throughput sequencing of DSB repair junctions confirm these results and shows significant depletion of indels with reduction in microhomology-mediated end-joining in AMBRA1-deficient cells, suggesting that AMBRA1 plays a role in promoting resection at DSBs. In support of this hypothesis, we found that the loss of AMBRA1 reduces RAD51 recruitment to DSBs in cells exposed to ionizing radiation. Consistent with its role in promoting HR, we found that AMBRA1-deficient cells are significantly more sensitive to PARP inhibitors compared to AMBRA1-proficient cells. In summary, we show that AMBRA1 is a novel regulator of DSBs, and that its downregulation in cancer cells and tumors render cancer cells susceptible to inhibition by PARP inhibitors.

Citation Format: Yuning Jiang, Tarek Abbas. Novel roles for AMBRA1 in regulating DNA double-strand breaks. [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 6107.
Abstract 6111: Design of a small molecule screening assay to detect DNA trapping of PARP1/2
Authors: Zientara-Rytter Kasia , Baron Veronique T. , Park Junguk , Shashkin Pavel , Zhu Henry
Published: April 2023

Poly(ADP-ribose) polymerase (PARP) inhibitors are currently used in the clinic for the treatment of tumors with a defective DNA damage response (DDR). When PARP1 or PARP2 binds damaged DNA, it adds poly(ADP-ribose) chains to its own backbone and to other DDR proteins, which recruits and activates them. PARylated PARP1/2 next detaches from the DNA so that the other PARylated proteins can initiate the repair process. It has been observed that some PARP inhibitors prevent PARP1/2 from dissociating the DNA. The continuous presence of PARP at the site of damage prevents repair and blocks replication, leading to cell death. Therefore, drugs that trap PARP1/2 to the DNA tend to be significantly more cytotoxic than other PARP inhibitors, which is highly desirable. This study describes the design and optimization of novel PARPtrap assays to specifically assess the ability of a drug to trap PARP onto DNA. The assay is based on principles of fluorescence polarization and uses fluorescently labeled DNA probes that are excited by polarized light and emit light with a degree of polarization that is proportional to the rate of molecular rotation. The free DNA probes that rotate fast have low fluorescence polarization (FP), but high FP when are bound to PARP1 or PARP2. When NAD+ is added, the PARylated enzymes detach from the probe, reducing FP levels. If a PARP inhibitor is added, the inhibitor’s trapping ability increases FP in a dose-dependent manner. Proof-of-principle titration of known PARP-trapping inhibitors (Talazoparib, AZD305, Olaparib and Veliparib) was performed to validate the assay. We observed that: i)The known relative trapping efficacies of Talazoparib, Olaparib and Veliparib were similar to known relative efficacies. ii)Talazoparib, Olaparib and Veliparib had similar trapping efficacy against PARP1 and PARP2, as measured by their EC50, whereas AZD305 was 1,000 times more efficient at trapping PARP1 than it was PARP2, demonstrating selectivity between PARP1 and PARP2. iii)AZD305 displayed as efficient DNA trapping activity toward PARP1 as best-in-class Talazoparib. In summary, we have designed an innovative PARPtrap assay designed for the high throughput screening of small molecule libraries to specifically identify or compare inhibitors that are capable of trapping PARP1 and/or PARP2 onto DNA.

Citation Format: Kasia Zientara-Rytter, Veronique T. Baron, Junguk Park, Pavel Shashkin, Henry Zhu. Design of a small molecule screening assay to detect DNA trapping of PARP1/2 [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 6111.
Abstract 6124: Recombinant apurinic/apyrimidinic endonuclease-1/redox factor-1 plus acetylsalicylic acid combination therapy inhibits tumorigenesis in an orthotopic xenograft murine model of triple-negative breast cancer
Authors: Lee Yu Ran , Joo Hee Kyoung , Lee Eun-Ok , Kim Sungmin , Jin Hao , Choi Yeon Hee , Choi Eun Ju , Jeon Byeong Hwa
Published: April 2023

Triple-negative breast cancer (TNBC) is characterized with hormone receptor negative and frequently exhibits inherently aggressive clinical behavior, resulting in poor prognoses. TNBCs are highly heterogenic, leading to obstacles in identifying new therapeutic targets and performing targeted therapy. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1), a multifunctional protein, is mainly involved in DNA repair and redox regulation; it is upregulated in various cancers and is extracellularly secreted because of hyperacetylation. We previously reported that hyperacetylation induced APE1/Ref-1 release causes apoptosis in TNBCs. In the current study, we aimed to assess the therapeutic efficacy of combination treatment involving recombinant human APE1/Ref-1 (rhAPE1/Ref-1) plus acetylsalicylic acid (ASA) in TNBC xenograft mice. ASA co-treatment was used to induce and lasting acetylation of rhAPE1/Ref-1. For in vivo analysis, we constructed an orthotopic xenograft in vivo model of TNBC by using MDA-MB-231 cells. We performed combination therapy involving intravenous injection of purified rhAPE1/Ref-1 (1 mg/kg) and oral administration of ASA (20 mg/kg) thrice a week for 6 weeks in TNBC xenograft mice. This rhAPE1/Ref-1 plus ASA combination therapy inhibited tumor formation and growth, without damaging liver or kidney tissue, and decreased plasma levels of the cancer marker CEA and breast cancer-specific markers CA27-29 and CA15-3 in the TNBC xenograft mice. The TNBC group, which underwent combination therapy, had more apoptotic cells and greater inflammatory cell infiltration in tumor tissues than the vehicle group did. Additionally, in tumor tissue of TNBC with combination therapy exhibits increased apoptosis following upregulation of Bax, cleaved-caspase-3, and cleaved-PARP. Thus, we found that rhAPE1/Ref-1 plus ASA combination therapy enhanced antitumor effects in TNBCs via inflammatory cell infiltration and apoptosis signaling pathways. Our findings indicate the therapeutic potential of rhAPE1/Ref-1 plus ASA combination therapy for TNBC.

Citation Format: Yu Ran Lee, Hee Kyoung Joo, Eun-Ok Lee, Sungmin Kim, Hao Jin, Yeon Hee Choi, Eun Ju Choi, Byeong Hwa Jeon. Recombinant apurinic/apyrimidinic endonuclease-1/redox factor-1 plus acetylsalicylic acid combination therapy inhibits tumorigenesis in an orthotopic xenograft murine model of 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 6124.
Abstract 6126: Antitumor effect of Iso-mukaadial acetate in MCF-7 human cancer xenograft models
Authors: Raphela-Choma Portia , Motadi Lesetja R. , Choene Mpho S.
Published: April 2023

Antitumor drugs used today have shown significant efficacy and are derived from natural products such as plants. Iso-mukaadial acetate has previously been shown to possess anticancer properties by inducing apoptosis. The purpose of this study was to investigate the therapeutic effect of Iso-mukaadial acetate in the breast cancer xenograft mice model. Female athymic mice were used and were inoculated with breast cancer cells subcutaneously after culturing. Group one served as a negative control group (no treatments) and group two positive control group (cisplatin) which was administered intravenously. Iso-mukaadial acetate treatment was administered orally to group three(100 mg/kg) and group four (500 mg/kg). Blood was collected (70 µL) from the tail vein on day zero, day seven and day 14. Tumor regression was measured, and a pharmacokinetic study was conducted. Estimation of Serum Parameters for Hepatic and Renal Indices were examined using an Automated Chemistry Analyzer. Histopathological analysis was conducted to evaluate morphological changes before and after treatment under a light microscope. Tumor shrinkage was observed after treatment. In conclusion, Iso-mukaadial acetate inhibited tumor progression in breast cancer athymic mice without major side effects.

Citation Format: Portia Raphela-Choma, Lesetja R. Motadi, Mpho S. Choene. Antitumor effect of Iso-mukaadial acetate in MCF-7 human cancer xenograft 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 6126.
Abstract 6145: Alternative splicing of GSDMB modulates killer lymphocyte-triggered pyroptosis
Authors: Kong Qing , Xia Shiyu , Pan Xingxin , Ye Kaixiong , Li Zhouyihan , Li Haoyan , Sahni Nidhi , Yi S. Stephen , Liu Xing , Wu Hao , Lieberman Judy , Zhang Zhibin
Published: April 2023

Killer lymphocyte-mediated tumor cell killing is a vital and final step of anti-tumor protective immunity. Killer cells, including cytotoxic CD8 T cells and Natural Killer (NK) cells, deliver granzymes (Gzm) into target cells, which normally activates noninflammatory cell death, but becomes inflammatory when target cells express the gasdermin (GSDM) proteins responsible for pyroptosis. GzmA and GzmB cleave and activate GSDMB and GSDME, respectively. However, the role of GSDMB in anti-tumor immunity is unclear - GSDMB pore formation is controversial and GSDMB expression has been linked to both anti- and pro-tumor functions. Here we found that GSDMB splicing variants are functionally distinct. Cleaved N-terminal (NT) fragments of isoforms 3 and 4 caused pyroptosis, but isoforms 1, 2 and 5, which deleted or modified exon 6, did not. Structural analysis based on AlphaFold predictions and the cryoEM structures of GSDMA3 and GSDMD showed that pore formation required the formation of a functional stabilizing belt structural element near the C-terminus of the cleaved N-terminal fragment, which was disrupted in the non-cytotoxic alternatively spliced isoforms. NK attack of GSDMB3-expressing cells caused pyroptosis, but GSDMB4 cells died mostly by apoptosis, and GSDMB1/2 cells died only by apoptosis. GSDMB4 partially resisted NK cell-triggered cleavage, suggesting that only GSDMB3 is fully functional. GSDMB1-3 were the most abundant isoforms in tested cells and were similarly induced by IFNγ and a chemotherapy drug methotrexate. Expression of cytotoxic GSDMB3/4, but not GSDMB1/2, was associated with better outcome in bladder and cervical cancer, suggesting GSDMB-mediated pyroptosis was protective in those tumors. Our study uncovers a novel role of alternative splicing in modulating caner-associated pyroptosis, and suggests that tumors may manipulate GSDMB splicing to avoid pyroptosis. Thus, modulating GSDMB splicing to increase cytotoxic GSDMB variants but suppress non-cytotoxic GSDMB isoforms could improve anti-tumor immunity and have therapeutic utility.

Citation Format: Qing Kong, Shiyu Xia, Xingxin Pan, Kaixiong Ye, Zhouyihan Li, Haoyan Li, Nidhi Sahni, S. Stephen Yi, Xing Liu, Hao Wu, Judy Lieberman, Zhibin Zhang. Alternative splicing of GSDMB modulates killer lymphocyte-triggered pyroptosis [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 6145.
Abstract 6146: Tumor suppressor p73 primes extrinsic apoptosis via transcriptional activation of DR5 and c-FLIP
Authors: Zhang Shengliang , El-Deiry Wafik S.
Published: April 2023

The tumor suppressor p73 is a member of p53 family. p73 transcriptionally activates p53-targeted genes involved in cell cycle regulation and apoptosis. It is well known that p73 induces cellular apoptosis via the intrinsic apoptotic pathway upon cellular stresses. However, the role of p73 in extrinsic apoptosis is poorly studied. We observed cell cycle arrested at the G1 phase in cells infected with a p73-expressing recombinant adenovirus (Ad-p73). We observed that overexpression of p73 upregulated c-FLIP at the protein level, in addition to DR5 and p21. c-FLIP is an anti-apoptotic factor that inhibits caspase 8/10 activation, thus blocking extrinsic pathway apoptosis. We demonstrated that c-FLIP is a transcriptional target of p73 based on our observations that: 1) p73 increases c-FLIP at mRNA level, 2) p73 binds to the c-FLIP promoter and 3) p73 enhances c-FLIP promoter-driven luciferase expression. We sought to explore whether the upregulation of c-FLIP prevents extrinsic apoptosis in cancer cells following p73 activation. To address this question, p73 was overexpressed in cells by adenovirus infection and c-FLIP was knocked down by siRNA. Overexpression of p73 induced cellular apoptosis in the cells with knockdown of c-FLIP and this effect was blocked by knockdown of caspase 8. Our results suggest that p73 induces extrinsic apoptosis when c-FLIP is depleted in cancer cells. We further found that the knockdown of DR5 inhibited p73-induced apoptosis in c-FLIP-deficient cancer cells. By contrast, PUMA knockdown did not block p73-induced apoptosis. Our results suggest that DR5 is required for cell extrinsic apoptosis in c-FLIP-deficient cancer cells upon p73 activation. p21 is the cyclin dependent kinase inhibitor that leads to cell cycle arrest at the G1/S phase. Knockdown of p21 enhanced cleaved-caspase 8 and cleaved-PARP in c-FLIP-depleted cancer cells treated with Ad-p73. Our results, taken together, suggest that the p73 primes extrinsic apoptosis via DR5 and the priming level appears to be titrated at the level of c-FLIP. Upregulation of c-FLIP decreases the priming level, resulting in cells converting to cell cycle arrest and survival via p21 whereas reduction of c-FLIP leads the primed cancer cells to undergo extrinsic apoptosis upon p73 treatment in cancer cells. Our discovery of p73 transcriptional upregulation of c-FLIP provides a rationale for depleting c-FLIP to improve antitumor efficacy of p73-targeting cancer therapy.

Citation Format: Shengliang Zhang, Wafik S. El-Deiry. Tumor suppressor p73 primes extrinsic apoptosis via transcriptional activation of DR5 and c-FLIP [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 6146.
Abstract 6149: STAT3 activation promotes invasiveness and cancer stemness in lung cancer
Authors: Yeung Sai Fung , Chan Sze Man , Law Chun Ho , Chan Hoi Yin , Tsui Kwok Wing
Published: April 2023

Lung cancer is the leading cause of cancer-related death worldwide, featuring a high incidence and low 5-year survival rate. Nearly 40% of patients are diagnosed with metastatic disease when they are first presented to the clinic. However, drug intervention targeting established metastatic tumors remains challenging, a better understanding of therapeutically exploitable mechanisms is urgently needed. This present study aims to explore novel therapeutic intervention against metastatic lung cancer. To understand the tumor-cell-intrinsic pathway that drives metastatic progression, we generated a highly invasive subline of lung cancer cell - A549 (ATCC, USA) by serial selections for Matrigel-coated transwells. The invasive subline A549-i3 shows about 5 times higher invasion potential than the parental cell line A549-Par (p<0.05, Student t-test). Colony-formation Assay and suspension growth in ultra-low attachment plates were used to measure the 2D proliferation ability and anoikis resistance, albeit with no difference. The differential efficacies between A549-Par and A549-i3 against multiple pathway inhibitors were screened using MTT assay in vitro. Notably, A549-i3 is more sensitive towards napabucasin (STAT3 inhibitor) and tipifarnib (FTase inhibitor) when compared with A549-Par, suggesting their potency in targeting cancer stemness and addicted oncogenic driver. Furthermore, napabucasin reduced the migration and invasion abilities of A549-i3 to similar extent as their parent counterpart. Overall, our preliminary data identified STAT3 as a potential therapeutic vulnerability in highly invasive cancer cells. Future work will be focused on identifying pathways responsible to sensitize highly invasive NSCLC cells toward STAT3 inhibition using multiomics approach. This project was supported by the Research Impact Fund (Ref. No.: R4015-19) from the Research Grants Council in Hong Kong.

Citation Format: Sai Fung Yeung, Sze Man Chan, Chun Ho Law, Hoi Yin Chan, Kwok Wing Tsui. STAT3 activation promotes invasiveness and cancer stemness in 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 6149.
Abstract 6164: NF-κB and NRF2 signaling pathways affect prognosis in HPV-associated head and neck cancer
Authors: Kothari Aditi , Schrank Travis Parke , Yarbrough Wendell Gray , Isaeva Natalia
Published: April 2023

Incidence of HPV associated head and neck squamous cell carcinoma (HNSCC) is on the rise, displaying a significantly favorable prognosis and overall better survival as compared to HPV negative HNSCC. Intense radiotherapy is the primary medical care leading to treatment associated morbidity in patients, thereby generating a need for de-intensification strategies. Biomarkers that can identify patients with less aggressive tumors as candidates for de-escalation therapy would revolutionize treatment. We applied an unbiased approach (weighted gene correlation network analysis (WGCNA)) that allows detection of autocorrelated gene sets on 3 independent cohorts of HPV+ HNSCC and created 22 consensus transcriptional modules by selecting genes that grouped together in WGCNA analyses from all 3 cohorts. Interestingly, only 1 module intrinsically divided HPV+ HNSCC into 2 subtypes, displaying different mutational profiles, mutational signatures, HPV gene expression patterns, HPV integration status, and patient survival. Gene set enrichment analysis revealed that this module was enriched in NF-kB genes and strongly associated with NF-κB signaling, confirming our prior findings that defined HPV+ HNSCC subtypes by presence or absence of NF-kB regulators, TRAF3 or CYLD defects and by the NF-κB activity classifier. Our studies show that NF- κB -driven intrinsic tumor characteristics contribute to increased sensitivity of NF- κB active HPV+ head and neck tumors to radiation, providing patients survival benefits. Indeed, TRAF3 or CYLD deletion activated NF-kB and dramatically increased radiation sensitivity of HPV+ head and neck cancer cells. In line with our RNA seq analysis, we found that activation of NF-κB via TRAF3/CYLD deletion significantly correlated with marked downregulation of NRF2 activity and reduced nuclear localization in in HPV+ HNSCC cells. Interestingly, TRAF3 CRISPR KO cells had lower NRF2 protein levels that were restored by MG132 treatment, indicating an involvement of KEAP1/CUL3 mediated proteasomal degradation of NRF2. In summary, our data unveils a unique relation between NF-κB pathway and radiosensitivity in HPV+ HNSCC. The distinct biological characteristics that separate patients into two subclasses associated with favorable prognosis could serve as a biomarker for personalized therapy.

Citation Format: Aditi Kothari, Travis Parke Schrank, Wendell Gray Yarbrough, Natalia Isaeva. NF-κB and NRF2 signaling pathways affect prognosis in HPV-associated head and neck 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 6164.