bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4288: Predicting DNA methylation in cervical cancer using somatic mutations in a classified mixed model prediction
Authors: Ramos Jairo D. , Rao J. Sunil
Published: April 2023

In cervical cancer, the promoter CpG islands of several tumor suppressor genes have been found with abnormal DNA methylation (DNAm) levels, which results in transcriptional silencing. Genetic databases from tumor samples are available without the corresponding DNAm profile. To fulfill this gap and contribute to deciphering the link between genetic variants and DNAm, we performed this study to find a good statistical model for predicting DNAm using genetic data. To address this, we downloaded from the TCGA dataset the genes with significant somatic mutations, as reported by the BROAD Institute, and the DNAm beta values, in known CpG sites, from 307 Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC) samples. Clinical data were also downloaded from patients, including race, age, gender, and cancer stage. We tested five mixed models containing both fixed and random effects to include the unmeasured source of variations in DNAm, for which we used the clustering of samples based on DNAm patterns. Models tested were: Classified mixed model prediction (CMMP), Linear regression model (LM), High dimensional shrinkage estimator based on the elastic net (ENET), a combination of CMMP - ENET, and Random Forest prediction (RF). Besides the significantly mutated genes, the clinical variables were also included as covariates to predict DNAm. We used shared random effects to borrow strength across racial groups, which improves predictive accuracy. Models were enhanced by combining other types of cancers to increase data heterogeneity and the number of samples. Lung Adenocarcinoma (LUAD) and Liver Hepatocellular Carcinoma (LIHC) were selected using Gini and Gap statistics for optimal clustering. Model fitting was evaluated using the average mean squared prediction error (MSPE). Results revealed that the CMMP model with CESC LIHC and LUAD data combined showed the best prediction results (lowest MSPE). The plot of the averaged estimated ENET coefficients indicated that the most important-significantly mutated genes to predict DNAm were CTNNB1, DMD, XIRP2, and PIK3CA. SNPs in these genes linked to cervical cancer include rs121913396, rs121913403 (CTNNB1), rs867262025, and rs121913279 (PIK3CA).In summary, we developed a mixed-effects model for accurately predicting DNAm levels in cervical cancer, using genes with significant somatic mutations (SNVs and INDELs). These results also align with growing evidence suggesting that genetic variation plays a role in DNAm.

Citation Format: Jairo D. Ramos, J. Sunil Rao. Predicting DNA methylation in cervical cancer using somatic mutations in a classified mixed model prediction. [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 4288.
Abstract 4292: Gene model correction for PVRIG and TIGIT in single cell sequencing data enables accurate detection and study of its functional relevance
Authors: Nemzer Sergey , Sabath Niv , Wool Assaf , Altber Zoya , Ando Hirofumi , Pardoll Drew M. , Ganguly Sudipto , Turpaz Yaron , Levine Zurit , Granit Roy Z.
Published: April 2023

Single cell RNA sequencing (scRNA-seq) has gained increased popularity in recent years and has revolutionized the study of cell populations; however, this technology presents several caveats regarding specific gene expression measurement. Here we examine the expression levels of several immune checkpoint genes, which are currently assessed in clinical studies. We find that unlike in most bulk sequencing studies, PVRIG and murine TIGIT suffers from poor detection in 10x Chromium scRNA-seq and other types of assays that utilize the GENCODE gene model. We show that the default GENCODE gene model, typically used in the analysis of such data, is incorrect in the PVRIG genomic region and contains also a predicted read-through transcript to which PVRIG reads co-align, causing these to be discarded and hence hindering its proper detection. Moreover, we find that the murine TIGIT 3’ UTR is mis-annotated, leading to the loss of legitimate reads. We thus generated a corrected reference genome, by removing the faulty read-through in the case of PVRIG and by extending the TIGIT 3’ UTR and demonstrate that by employing these changes we can correctly capture genuine expression levels of these checkpoints, and which align with our findings at the protein level using FACS and CITEseq. Furthermore, we show that specialized read multimap algorithms such as RSEM and STARsolo can also partially improve the detection of PVRIG. Our study provides means to better interrogate the expression of PVRIG and murine TIGIT in scRNA-seq and emphasize the importance of optimizing gene models and alignment algorithms to enable accurate gene expression measurement in scRNA-seq and bulk sequencing. Moreover, our results support detailed study of the expression of immune checkpoints in clinical and pre-clinical studies towards the development of cancer immunotherapy treatments.

Citation Format: Sergey Nemzer, Niv Sabath, Assaf Wool, Zoya Altber, Hirofumi Ando, Drew M. Pardoll, Sudipto Ganguly, Yaron Turpaz, Zurit Levine, Roy Z. Granit. Gene model correction for PVRIG and TIGIT in single cell sequencing data enables accurate detection and study of its functional relevance. [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 4292.
Abstract 4293: Strategies and resources for applying a quantitative multiplex IHC imaging workflow to characterize immune contexture in solid tumors
Authors: Sivagnanam Shamilene , Betts Courtney M. , Kirchberger Nell , Betre Konjit , Coussens Lisa M.
Published: April 2023

Conventional immunohistochemistry (IHC) is a standardized diagnostic technique used in tissue pathology. However, the capacity to label only one marker per tissue section is a critical limitation. Multiplex immunohistochemistry (mIHC) is a powerful imaging technique used in basic, translational research and clinical settings to simultaneously detect the expression of multiple epitopes in a single formalin-fixed paraffin-embedded (FFPE) tissue section, allowing for characterization and quantification of cells while maintaining their spatial location. This technique allows for a comprehensive view of the immune milieu, expression patterns, and interactions between cell types that can help elucidate the complexity of the tumor-immune microenvironment (TiME). Multiplex imaging has emerged as a powerful tool to better understand tumor progression, response or resistance to therapy, and potentially identify predictive biomarkers. However, this technique requires careful tissue considerations and generates enormous amounts of hierarchical data, including single cell marker expressions, locations, and shape features, resulting in complex and challenging analyses. We previously published a validated platform using nine matched primary and recurrent head and neck squamous cell carcinoma (HNSCC) sections stained sequentially with a panel of 29 antibodies identifying malignant tumor cells, and 17 distinct leukocyte lineages and their functional states1. Here, we have optimized and consolidated important considerations, including tissue quality and QC steps necessary for generating high quality data from multiplex imaging studies. We demonstrate strategies for a streamline analytic pipeline using our quantitative workflow developed to characterize immune cells and infiltration patterns within spatial compartments of solid tumors in FFPE tissues, and herein, provide strategies and resources to practically adapt and utilize our analytic mIHC pipeline.

1. Banik, G., et al., Methods Enzymol., 2020

Citation Format: Shamilene Sivagnanam, Courtney M. Betts, Nell Kirchberger, Konjit Betre, Lisa M. Coussens. Strategies and resources for applying a quantitative multiplex IHC imaging workflow to characterize immune contexture in solid tumors. [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 4293.
Abstract 4347: A pivotal clinical trial of cResponse, a functional assay for cancer precision medicine
Authors: Salpeter Seth , Bar Vered , Neev Guy , Zundelevich Adi , Rosen Gil , Hanks Sandra , Costelloe Naoise , Krell Jonathan , Straussman Ravid
Published: April 2023

Precision cancer therapy has the potential to revolutionize treatment outcome. While genomic analysis has become central to cancer personalized medicine, recent studies have not shown that it drastically improves the patient’s survival as compared to standard drug selection. Additionally, genomic mutations may suggest several treatment protocols without elucidating which approach will yield the best clinical response. To advance cancer precision guidance, we have developed cResponse, a combined genomic-functional drug sensitivity platform to determine individualized patient treatment regimens. Fresh patient cancer samples are taken by biopsy or resection and sectioned into 250uM slices which when cultured in cResponse platform demonstrate similar architecture and tissue proliferation to those found in vivo. An initial clinical study showed that cResponse can preserve human cancer tissue in 3D culture together with its microenvironment, including endothelial and immune cells, at a high viability (>90%) with continued cell division for more than 7 days. On a cohort of 34 patients treated with neoadjuvant therapy or systemic therapy for metastatic disease, the assay was able to predict patient response to drug treatment with a sensitivity of 96% and a specificity of 77.7%. To further validate the capacity of the cResponse platform to predict patient response to cancer therapy, a follow up pivotal clinical study was established at 7 major cancer centers located in the UK with the goal of recruiting a total of 170 patients to provide a large statistical validation of the previous results. Here we report on the outcome of the first 50 patients recruited to the pivotal trial and describe the predictive results correlated to patient response.

Citation Format: Seth Salpeter, Vered Bar, Guy Neev, Adi Zundelevich, Gil Rosen, Sandra Hanks, Naoise Costelloe, Jonathan Krell, Ravid Straussman. A pivotal clinical trial of cResponse, a functional assay for cancer precision medicine. [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 4347.
Abstract 4386: Comparison of cytotoxicity of anthracycline based antineoplastic drugs in breast cancer
Authors: Singhal Mitali , Gomez Jacobo Elies , Nayak Sanjit , Suman Kirsten Riches , Estrada Amalia Ruiz
Published: April 2023

Mitoxantrone (MTX) is used for the chemotherapeutic treatment of breast cancer, but it has a dose-limiting cardiotoxicity. One of the mechanisms of MTX cytotoxicity is by inhibiting the topoisomerase II enzyme, which is crucial for maintaining cellular processes like replication. The aim of the study is to investigate if an alternative analog of MTX, named KP71 would be a suitable antineoplastic drug with reduced cardiovascular side effects. Cytotoxicity of both drugs in breast cancer cell lines MDA-MB-468, MDA-MB-231, MCF7, the non-neoplastic breast cell line MCF10, cardiac human fibroblasts (HFB), and human umbilical vein endothelial cells (HUVEC) was assessed by MTT at 96h. The IC50 values (nM) for MTX/KP71 were as shown in table below. DNA decatenation assay demonstrated topoisomerase II inhibition with values of IC50 = 2.9/12.3 µM for topoisomerase IIα, and 2.1/7.0 µM for topoisomerase IIβ for MTX/KP71 respectively. Western blotting showed DNA damage by the gradation in γH2AX expression at different concentrations of MTX and KP71 at 6 h and 24 h treatment. KP71 was found to retain the ability to inhibit the proliferation of neoplastic cell lines in vitro with a slower profile of cytotoxicity on non-neoplastic cell lines as compared to MTX. KP71 partly exerted cytotoxicity by damaging DNA and inhibiting topoisomerase II enzyme activity. Organotypic HFB-HUVEC co-cultures were used to investigate the effects of anthracycline drugs in angiogenesis. Although both drugs reduced the number of tubules (and branches) compared to control (absence of drug), there were not significant differences between MTX and KP71.

MTT IC50 (nM) MTX KP71 MDA-MB-468 27 50 MDA-MB231 38 169 MCF7 63 150 MCF10 35 90 HFB 160 1177 HUVEC 56 93

Citation Format: Mitali Singhal, Jacobo Elies Gomez, Sanjit Nayak, Kirsten Riches Suman, Amalia Ruiz Estrada. Comparison of cytotoxicity of anthracycline based antineoplastic drugs in 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 4386.
Abstract 45: Elephant p53 protects mice from carcinogen induced death
Authors: Abegglen Lisa M. , Fowles Jared S. , Preston Aidan J. , Rogers Aaron , Bhachech Niraja , Barney Brayden B. , Kennington Ryan , Lum David H. , Mitchell Gareth , Schiffman Joshua D.
Published: April 2023

Elephants naturally have low rates of cancer, potentially due to evolved genetic changes in tumor suppressor elephant TP53 (EP53) and amplification of 19 TP53 retrogenes. A better understanding of the mechanisms of cancer suppression in elephants by EP53 and its retrogenes could lead to more effective human cancer therapeutics. EP53 induces a strong apoptotic response compared to human TP53, especially when combined with EP53-RETROGENE 9 (EP53-R9). EP53-R9 encodes a truncated p53 protein that induces apoptosis of human cancer cells independent of EP53 through a transcription-independent mechanism. To characterize EP53 and EP53-R9’s role in cancer suppression, transgenic mice were generated to replace mouse TRP53 with EP53. Additionally, a tetracycline inducible EP53-R9 gene was inserted into a safe harbor locus in mice. Carcinogenesis studies with 3-Methylcholanthene injection revealed that EP53 mice survived significantly longer compared to heterozygous or homozygousTRP53 mice (p <0.0001, 0.0102). Experiments to assess the protective role of EP53-R9 alone and combined with EP53 are ongoing. Several mouse embryonic fibroblast (MEF) cell lines were generated from these transgenic mice with 14 distinct genotypes with different combinations of TRP53, EP53, and EP53-R9. p53 target gene expression studies showed that MEFs with EP53 induce higher expression of MDM2 and p21 compared to TRP53-containing MEFs, suggesting that the cancer-protective effect observed in EP53 mice is due, in part, to greater activation of p53 signaling. These results support the need for future work to assess the potential of EP53-based therapeutics for human cancer.

Citation Format: Lisa M. Abegglen, Jared S. Fowles, Aidan J. Preston, Aaron Rogers, Niraja Bhachech, Brayden B. Barney, Ryan Kennington, David H. Lum, Gareth Mitchell, Joshua D. Schiffman. Elephant p53 protects mice from carcinogen induced death [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 45.
Abstract 4517: Novel prognostic and predictive biomarker for neuroblastoma
Authors: Somasundaram Dinesh Babu , Yu Zhongxin , Baker Ashley , Aravindan Natarajan
Published: April 2023

Recently we showed that Retinal degeneration protein 3 (RD3) is constitutively expressed in healthy adult and fetal tissues beyond retina and, its gradient expression from high to low in ganglioneuroma, ganglioneuroblastoma and neuroblastoma (NB). Crucially, our sequential in vitro and in vivo studies identified that RD3 regulates NB progression and metastasis, and its loss prompts the evolution of therapy defying progressive disease (PD). Here we investigated the prognostic significance of RD3 in NB, its predictive benefit and, its association to clinical outcomes. RNA-Seq data mining (4 independent study cohorts, total n = 1013) indicated that high RD3 can predict good NB prognosis. RD3 expression (immunohistochemistry) gauged in our NB patient cohort (n = 100) showed a positive correlation with longer overall survival (OS, P = 1.0E-04) and relapse-free survival (RFS, P = 2.1E-02). Similarly, high RD3 correlated with longer OS (P = 1.2E-03) and RFS (P = 2.1-02) in children presented with high-risk NB (HR-NB), irrespective of MYCN amplification status (MYCN amplified, P = 1.4E-02; non-amplified, P = 5.5E-03) and patient age (<2Y age, P = 2.8E-02; >2Y age, P = 1.0E-04) groups. Significantly, we observed a gradient loss of RD3 with NB progression, from stage 1 through stage 4 disease. Consistently, RD3 loss was substantial in metastatic disease when compared to the primary neuroblastomas, and this loss associated with poor OS in patients presented with metastatic disease (P = 4.0E-02). More importantly, RD3 is significantly lost in therapy resistant progressive NB and corroborated significantly (P = 9.5E-03) with the poor progression free survival. Multivariate analysis identified RD3 as an independent (P = 4.4E-02) prognostic factor for NB. These results demonstrate that RD3-loss can forecast poor prognosis in NB, and further imply that assessing RD3 loss could serve as the good predictor for therapy response and/or PD evolution. Together, these data identify a novel prognostic and predictive factor for neuroblastoma. Funding: This work was partially or in full, funded by Oklahoma Center for the Advancement of Science and Technology, OCAST-HR19-045; National Institutes of Health, NIH-P20GM103639 and Department of Defense DoD-CA210339

Citation Format: Dinesh Babu Somasundaram, Zhongxin Yu, Ashley Baker, Natarajan Aravindan. Novel prognostic and predictive biomarker for neuroblastoma. [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 4517.
Abstract 4554: Head and neck cancer HuBiogel-embedded microtumor assay system for therapeutic efficacy testing of patient tumor specimens
Authors: Willey Christopher D. , Ying Yedeh P. , Morlandt Anthony B. , Amm Hope M. , Hicks Patricia H. , Anderson Joshua C. , Beierle Andee M. , Thomas Carissa M. , Warram Jason M. , Chen Jingsong , Thomas Jeffrey A. , Banko Katie , Singh Raj K.
Published: April 2023

Head and neck (HN) cancer recurrence is common, and selecting effective salvage systemic therapy remains difficult, particularly for oral cavity cancers. Developing a rapid, robust and predictive therapeutic testing system could support clinical decision-making and improve patient outcomes. We developed a Patient Therapy Evaluation System (PTES) that employs a three-dimensional (3D) fully human microtumor drug testing assay using tissue specimens collected during surgery of HN cancers. Remnant fresh tumor tissue from patients is dissociated into single-cell suspension that is embedded using a novel HuBiogel-cell encapsulation technology (3D microtumors). This high-throughput assay platform allows morphologic, functional, and molecular evaluations in parallel by real-time imaging, cell proliferation, and biomarker protocols. Microtumor viability, growth profiles, and drug screening data are captured at multiple time points up to 14 days. Our initial cohort included 57 patient specimens (53 squamous cell carcinomas, 1 verrucous carcinoma, 1 osteosarcoma, 1 ameloblastoma, and 1 non-cancerous lichenoid mucositis). HuBiogel-embedded tumor cells formed numerous multicellular colonies exhibiting distinct organization and growth patterns in 14-day microtumor cultures. Interestingly, epithelial, stromal and stem-cell like populations were preserved in HN microtumor models based on marker expression. Treatment with single (cisplatin, 5FU, docetaxel) drugs and their combinations resulted in tumor inhibitory responses (IC50) evaluated by CellTiter-Glo assay, and residual surviving cells were also recorded by Calcein-AM staining of 3D Microtumors. While patient-derived HN microtumors were produced with high success rates, factors associated with lower microtumor yield included smaller tumor specimens and low viability after dissociation. In conclusion, our new all human microtumor assay models replicating phenotypic, functional, and molecular properties ex vivo provide a potential theranostic tool for rapidly predicting drug sensitivity and improving treatment strategy for HN cancer patients.

Citation Format: Christopher D. Willey, Yedeh P. Ying, Anthony B. Morlandt, Hope M. Amm, Patricia H. Hicks, Joshua C. Anderson, Andee M. Beierle, Carissa M. Thomas, Jason M. Warram, Jingsong Chen, Jeffrey A. Thomas, Katie Banko, Raj K. Singh. Head and neck cancer HuBiogel-embedded microtumor assay system for therapeutic efficacy testing of patient tumor specimens. [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 4554.
Abstract 4718: Analyses of epigenetic marks and mechanisms in disease: Your guide to a successful CUT&RUN assay
Authors: Guo Angela H. , Comeau Christopher R. , Chen Fang , Fry Christopher J.
Published: April 2023

Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets & Release Using Nuclease (CUT&RUN) is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell. The CUT&RUN assay can be combined with downstream qPCR or NG-seq to analyze histone modifications and binding of transcription factors, DNA replication factors, or DNA repair proteins at specific target genes or across the entire genome. CUT&RUN provides a rapid, robust, and true low cell number assay for detection of protein-DNA interactions in the cell. Unlike the ChIP assay, CUT&RUN is free from formaldehyde cross-linking, chromatin fragmentation, and immunoprecipitation. Previously, we have shown that, compared to ChIP, CUT&RUN requires fewer starting cells (100K), has a much faster protocol (one day from cells to DNA), generates lower background signal (requires less sequencing depth), and offers spike-in control DNA for effective normalization of signal between samples and between experiments. We recently updated the CST CUT&RUN Assay Kit for use with 5,000-20,000 cells and added protocols for fixed cells and tissue. I will discuss the basics of the CUT&RUN assay and important factors to consider when setting up your experiment. In addition, I will provide data showing the versatility of this assay for mapping various histone modifications, transcription factor, and transcription cofactor binding across multiple sample types. Finally, I will discuss how the general protocol is optimized for greater signal to noise ratio, reduced number of starting cells, and provide an alternative digestion method to prepare the input DNA as a critical control of the CUT&RUN experiment.

Citation Format: Angela H. Guo, Christopher R. Comeau, Fang Chen, Christopher J. Fry. Analyses of epigenetic marks and mechanisms in disease: Your guide to a successful CUT&RUN assay. [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 4718.
Abstract 4719: Alterations of chromatin structure and TGF-β signaling in ARID1A-mutated colon cancer cells
Authors: Kim Jiwon , Jang Giyong , Lee Soo-Jin , Lee Sang Eun , Cho Sung-Yup
Published: April 2023

ARID1A, a subunit of the SWI/SNF complex which regulates gene expression by remodeling chromatin structure, is often mutated in various cancer and is known as a tumor suppressor gene. Many studies revealed the loss of ARID1A promotes tumor progression by activating PI3K-Akt signaling, but other pathways have not yet been fully elucidated. By analyzing RNA sequencing data and ATAC-seq data with ARID1A wild-type cell lines HCT116 and isogenic ARID1A Q456*/Q456* which has truncation mutations for ARID1A, we investigated the alterations of epigenomic and transcriptomic characteristics according to the loss of ARID1A. The overall openness of chromatin decreased in ARID1A Q456*/Q456* cells. Since SWI/SNF complex regulates chromatin structure and gene expression accordingly, we analyzed genes whose change of RNA expression and openness of chromatin in the same direction. The results showed 109 upregulated genes and 715 downregulated genes compared to wild type cell line with Padj < 0.05, |log2FC| > 0.58 from both RNA sequencing and ATAC-seq. To determine which gene sets are activated in the ARID1A-deficient cell line, the differentially expressed genes with consistent chromatin openness were subjected to Gene Ontology analysis. The upregulated genes in the ARID1A-deficient cell line were enriched in ‘Cell proliferation’ and ‘TGF-β signaling’. In addition, the expression of representative genes associated with TGF-β signaling increased in ARID1A-deficient cells. Our data suggests that loss-of-mutation in ARID1A significantly changes the chromatin structure, resulting in alterations of gene expression associated with cell proliferation and TGF-β signaling.

Citation Format: Jiwon Kim, Giyong Jang, Soo-Jin Lee, Sang Eun Lee, Sung-Yup Cho. Alterations of chromatin structure and TGF-β signaling in ARID1A-mutated colon cancer cells. [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 4719.
Abstract 4721: Yin Yang 1-induced long non-coding RNA DUXAP9 drives oral squamous cell carcinoma by blocking CDK1-mediated EZH2 degradation
Authors: Cao Wei , Zhou Wenkai
Published: April 2023

Background: lncRNAs play a critical role in oral squamous cell carcinoma (OSCC) progression and metastasis. However, the detailed molecular mechanism is not fully understood.

Methods: Differential-expressed lncRNAs between OSCCs and paired adjacent tissues were identified by microarray. The expression of lncRNADUXAP9 (DUXAP9) was detected by RNAscope and quantitative real-time PCR (qRT-PCR). The gain-of-function and loss-of-function of DUXAP9 were evaluated in vitro and in vivo assays. Chromatin immunoprecipitation (ChIP)and luciferase assays were performed to analyze the transcriptional regulation of DUXAP9. RNA pull-down assays, LS-MS/MS analysis, RNA immunoprecipitation assays, and Co-immunoprecipitation (Co-IP) were performed to address the molecular mechanism through which DUXAP9promotes OSCC progression.

Results: By analyzing lncRNA microarray data, we identified a novel nuclear-localized DUXAP9, which is upregulated in OSCC. The high level of DUXAP9is positively associated with lymph-node metastasis, poor pathological differentiation, advanced clinical stage, worse overall survival, and worse disease-specific survival in OSCC patients. DUXAP9 significantly promotes cell proliferation, migration/invasion, and metastasis in vitro and in vivo through an EZH2-dependent manner. Yin Yang 1(YY1) was found to activate the transcriptional expression of DUXAP9 in OSCC. Furthermore, DUXAP9was proven to physically interact with EZH2 and inhibit EZH2 degradation via the suppression of the phosphorylation of EZH2, thereby blocking EZH2nucleus to cytoplasm translocation.

Conclusions: Here we demonstrated a novel mechanism mediated by lncRNA DUXAP9 at the posttranslational level, which contributes to OSCC progression, thus providing a promising target for OSCC therapy.

Keywords: LncRNA DUXAP9, YY1, EZH2, Ubiquitination, Oral squamous cell carcinoma

Citation Format: Wei Cao, Wenkai Zhou. Yin Yang 1-induced long non-coding RNA DUXAP9 drives oral squamous cell carcinoma by blocking CDK1-mediated EZH2 degradation. [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 4721.
Abstract 4726: KMT5C-dependent regulation of mesenchymal-related genes and identification of KMT5C interactome using BioID
Authors: Son Jihye , Agredo Alejandra , Glaws Ally , Kasinski Andrea L.
Published: April 2023

Epigenetic dysregulation has recently been recognized as a new hallmark of cancer. Dysregulation of chromatin remodelers is estimated to affect 10-20% of cancer. Thus, understanding how epigenetic-based mechanisms contribute to cancer initiation and progression is critical. KMT5C is a histone methyltransferase that catalyzes histone H4 lysine 20 trimethylation (H4K20me3), which has historically been associated with formation and maintenance of constitutive heterochromatin regions. Albeit recent evidence from our lab also suggests regulation of gene-rich euchromatin regions. In this previous body of work we discovered that loss of KMT5C promotes development of resistance to tyrosine kinase inhibitors (TKI) in EGFR-mutant non-small cell lung cancer (NSCLC) cells (PC9 and HCC827). Patient data further supports that the KMT5C transcript level is downregulated following resistance to osimertinib, a third-generation TKI. Our data suggests that KMT5C is involved in non-canonical regulation of genes outside of heterochromatic regions through its methyltransferase activity. Analysis of data deposited into the Cell Miner database and publicly available RNA-sequencing and ChIP-sequencing data revealed that the KMT5C-H4K20me3 axis has significant negative correlations with several genes involved in epithelial-to-mesenchymal transition (EMT). Our validation studies support this observation. However the mechanisms involved in KMT5C facilitating this change at EMT-related genes is unknown. KMT5C is well understood to be recruited to heterochromatin regions through its interaction with HP1, yet the mechanism for recruitment of KMT5C to gene-rich euchromatin regions remains to be elucidated. We hypothesize that KMT5C is recruited to its target genes in euchromatin in an HP1-independent mechanism. In this study, we aim to identify the interactome of KMT5C using an unbiased proximity labeling BioID approach. This study is expected to uncover a novel epigenetic mechanism that contributes to development of resistance to TKIs and may inform of strategies to target KMT5C-downregulated TKI-resistant cancers.

Citation Format: Jihye Son, Alejandra Agredo, Ally Glaws, Andrea L. Kasinski. KMT5C-dependent regulation of mesenchymal-related genes and identification of KMT5C interactome using BioID. [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 4726.
Abstract 4796: Chemotherapy induced senescence drives peripheral neuropathy
Authors: Malachowski Taylor , Raut Ganesh , Bagchi Satarupa Mullick , Stewart Shelia
Published: April 2023

Chemotherapy is a mainstay of cancer therapy. Unfortunately, while chemotherapy can profoundly impact disease free survival, it’s often accompanied with devastating side effects, including peripheral neuropathy. Indeed, 30-40% of patients treated with neurotoxic chemotherapy develop long-term and often debilitating chemotherapy-induced peripheral neuropathy (CIPN). Unfortunately, there are currently no preventative measures for CIPN and while it is transitory in some patients, for others the side effects can persist for months or even years after the cessation of chemotherapy. Recent work suggests that cellular senescence, which is robustly induced by chemotherapy, contributes to CIPN. Senescent cells are typically characterized by increased CDKn2a (i.e., p16) expression, increased SA-β-gal hydrolyzation, and expression of the senescence-associated secretory phenotype (SASP) that can influence multiple cell types in the microenvironment. Through utilization of a mouse model that employs paclitaxel (PTX), we find that PTX robustly induces senescence in the hindpaws and dorsal root ganglia (DRG) of mice that display loss of peripheral axons and decreased response to mechanical stimuli. To address the role of senescence in CIPN, we utilized the INKATTAC mouse that allows for inducible elimination senescent cells. Using this model, we find that the elimination of senescent cells rescues CIPN. Further, the use of senolytics, drugs that kill senescent cells, also rescues CIPN, raising the possibility that we can treat patients with CIPN. To address the mechanism behind CIPN we have carried out single cell RNA-Seq to identify the population of senescent cells senescing in response to chemotherapy. These analyses will allow us to understand the mechanisms that drive CIPN and may lead to new treatments for patients suffering from CIPN.

Citation Format: Taylor Malachowski, Ganesh Raut, Satarupa Mullick Bagchi, Shelia Stewart. Chemotherapy induced senescence drives peripheral neuropathy. [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 4796.
Abstract 4816: Targeting GCN2 regulation of amino acid homeostasis sensitizes prostate cancer cells to senolytic therapies
Authors: Sommers Noah R. , Cordova Ricardo A. , Klunk Angela J. , Pili Roberto , Wek Ronald C. , Staschke Kirk A.
Published: April 2023

The integrated stress response (ISR) features a family of eIF2 kinases that sense cellular stress, triggering translational and transcriptional modes of gene expression that enhance cell adaptation to the underlying stress. Previously we reported (Cordova et al., 2022 eLife) that prostate cancer (PCa) cells rely on one of these eIF2 kinases, GCN2, for maintenance of amino acid homeostasis and sustained proliferation. GCN2 functions to enhance expression of amino acid transporters and subsequent import of essential amino acids (EAAs) that are necessary to sustain PCa growth. Genetic loss or pharmacological inhibition of GCN2 results in decreased expression of amino acid transporters, severe depletion of intracellular EAAs, and decreased proliferation of PCa cells in culture and mouse xenograft models. Although loss of GCN2 in PCa cells reduces proliferation, depletion of this eIF2 kinase is suggested to lead to cell stasis and modest cell death. We hypothesize that the static phenotype by GCN2 inhibition in PCa cells renders these cells vulnerable to senolytic treatment and is a potential therapeutic target in combination therapies. To address this idea, we utilized models of androgen-sensitive and castration-resistant PCa and non-tumorigenic prostate epithelial cells. GCN2 inhibition induced a static phenotype in PCa cell lines, but not non-tumorigenic prostate epithelial cells, and this was accompanied by increased expression of the senescent markers p53, p21, p27, and MCL-1. Furthermore, induction of senescent markers following GCN2 inhibition in PCa cells was reversed by supplementation with EAAs, suggesting that limitation of EAAs is critical to the induction of cell stasis. Of importance, treatment of PCa cells with Navitoclax, a senolytic agent that targets Bcl-2 family of anti-apoptotic proteins, in combination with GCN2 inhibition resulted in enhanced cell death and apoptosis compared to each single treatment alone. Our results suggest that GCN2 inhibition in PCa cells leads to depletion of EAAs and induction of a static phenotype, rendering these cells vulnerable to targeting the Bcl-2 family of proteins to expedite cell death. As such, therapies integrating GCN2 inhibition and senolytic agents are proposed to be effective strategies for treatment of PCa.

Citation Format: Noah R. Sommers, Ricardo A. Cordova, Angela J. Klunk, Roberto Pili, Ronald C. Wek, Kirk A. Staschke. Targeting GCN2 regulation of amino acid homeostasis sensitizes prostate cancer cells to senolytic therapies. [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 4816.
Abstract 4819: Mechanism of disturbance of nuclear size homeostasis in cancer cells
Authors: Kim Changgon , Kim Joon
Published: April 2023

Abnormal increase in nuclear size is one of the criteria for distinguishing cancer cells. The main causes and functional consequences of nuclear size increase in cancer cells are not well known. In this study, we analyzed genome-wide RNAi library screen data to discover genes involved in the maintenance of nuclear size homeostasis. Importantly, many of the screen hits mapped to the functional categories of replication stress and DNA damage repair. We confirmed that depletion of genes required to prevent replication fork collapse causes a significant increase in nuclear size. Excessive replication stress and defects in the DNA repair system are common in many cancers. Therefore, disturbances of nuclear size homeostasis may be due to excessive replication stress and enhanced DNA damage response. To identify effectors that link replication stress and nuclear size change, we investigated candidates such as cell cycle progression, the mTOR pathway, and the cytoskeleton. As previously known, cell cycle arrest at G2 phase increased nuclear size, but replication fork collapse increased nuclear size even more excessively, suggesting the existence of other mechanisms. Inhibition of the mTOR pathway did not block replication stress-induced nuclear enlargement. Interestingly, we found that actin polymerization inside the nucleus plays a role in determining nuclear size in replication stress condition. Inhibition of nuclear actin polymerization reversed nuclear enlargement due to replication fork collapses. The Increase in nuclear size appeared to reduce the invasive ability of cancer cells. In addition, the enlarged nuclei resisted mechanical stress-mediated nuclear envelope rupture. We also observed a change in the abundance of the tri-methylation of lysine 27 on histone H3. These results suggest that nuclear size homeostasis is perturbed as a result of excessive replication stress, and changes in nuclear size affect the invasive capacity and gene expression of cancer cells. However, it is not clear whether nuclear enlargement act as a strong barrier to metastasis, as nuclear size can readily change through changes in nuclear actin dynamics. Further studies are needed to understand the impact of nuclear size changes on cancer initiation and progression.

Citation Format: Changgon Kim, Joon Kim. Mechanism of disturbance of nuclear size homeostasis in cancer cells. [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 4819.
Abstract 4821: Exploring the role of a solid-like condensate in breast and prostate cancer
Authors: Grunfeld Alexander , Bokros Michael , Kirk Chloe , Lee Stephen
Published: April 2023

Cancer cells are exposed to harsh environmental conditions, which they adapt to by engaging various stress response pathways. A common cellular response to these stressors is the assembly of membraneless compartments known as biomolecular condensates. Our lab discovered one such pathway where exposure to environmental stressors reversibly converts nucleoli into solid-like condensates known as Amyloid bodies (A-bodies). A-bodies induce a state of cellular dormancy by sequestering in an amyloid-like state key proteins involved in processes such as DNA replication and cell cycle progression. In vitro experiments showed that suppressing A-body formation in cancer cell lines accelerates tumorigenesis, suggesting that system-wide amyloidogenesis is a mechanism of tumor suppression. The goal of this project was to investigate how A-bodies correlate with tumorigenesis in the context of human tissues. Using formalin-fixed paraffin embedded (FFPE) sections of breast invasive ductal carcinoma and prostate adenocarcinoma, we first confirmed the presence of A-bodies in tumors by staining with the amyloidophilic dye Amylo-glo. In agreement with in vitro data in breast and prostate cancer cell lines, immunohistochemical staining further showed these condensates contain known A-body constituent proteins. Co-staining tissues with Amylo-glo and the proliferation marker Ki67 revealed a negative correlation between A-bodies and cell proliferation, supporting the hypothesis that A-bodies suppress cell growth. Finally, tissue staining comparing primary tumors with distant metastases suggests that A-body biogenesis is reduced in the progression to metastatic disease. Together, our preliminary data presents a biomolecular condensate which can be visualized in human tissues and inversely correlates with cell proliferation. Future directions include examining additional tumor types for the presence of A-bodies and testing the correlation between A-bodies and other markers of proliferation, cell cycle arrest, and environmental stress.

Citation Format: Alexander Grunfeld, Michael Bokros, Chloe Kirk, Stephen Lee. Exploring the role of a solid-like condensate in breast and 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 4821.
Abstract 4932: The role of KB-S in treating pancreatic cancer
Authors: Boshra Sadia Noosrat , Bisht Kirpal , Acevedo-Duncan Mildred
Published: April 2023

Pancreatic cancer remains one of the most lethal malignant cancers because the symptoms of this cancer do not exhibit until the disease progresses to an advanced stage. Late diagnosis may result in metastasis. When metastasis occurs, cancer can no longer be removed with surgery and therefore, requires treatment with FDA approved chemotherapeutic drugs or radiotherapy and they often fail to treat the disease. KB-S is a synthetic thymidine nucleoside analogue which is broadly used for the treatment of Human Immunodeficiency Virus (HIV). In this work, we explored the therapeutic potential of KB-S as an anticancer drug, with focused against pancreatic cancer. Our aim is to establish a connection between cellular kinase of KB-S and atypical protein kinase C (aPKC) to decipher signaling pathways. Pancreatic adenocarcinoma, ASPC-1 cell line was used for the study. Hemocytometer was used for cell counting for the dose response curve after treating with KB-S. Immunoprecipitation study was conducted using anti-PKC-iota beads. Western blot assay was performed using primary antibody PKC-zeta and a secondary antibody. Our results showed that KB-S decreases cell proliferation significantly on AsPC-1 cells over increasing the concentrations. KB-S decreased the proliferation by 20% for 0.5 µM (P≤ 0.09), 37% for 5 µM (P≤ 0.02), 44% for 10 µM (P≤ 0.01), 48% for 20 µM (P≤ 0.002) and 54% for 50 µM (P≤ 0.001) in AsPC-1 cells. We used the lowest concentration of 10 µM with significant inhibition for further experiments. We immunoprecipitated for PKC-ι and immunoblotted for PKC-ζ and the immunoblots showed that there is no association between PKC-ι and PKC-ζ in pancreatic cancer cell line (AsPC-1). But when we performed reverse immunoprecipitation, we found that there is an association between PKC-ι and PKC-ζ. Again, we immunoprecipitated for thymidine kinase and immunoblotted for PKC-ι and immunoblots showed that there is an association between thymidine kinase and PKC-ι. But when we did the reverse immunoprecipitation, we did not find any association between thymidine kinase and PKC-ι in pancreatic cancer cell line. Based on our preliminary studies, we report the anti-cancer effect of KB-S in treating a pancreatic cancer cell line leading to further future investigations. To the best of our knowledge, this is the first demonstration of therapeutic efficacy of KB-S against a cancer. Given that, KB-S is already approved by FDA for the treatment of HIV, it carries the potential to be repurposed for difficult to cure cancerous diseases such as pancreatic cancer.

Citation Format: Sadia Noosrat Boshra, Kirpal Bisht, Mildred Acevedo-Duncan. The role of KB-S in treating pancreatic 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 4932.
Abstract 4962: Discovery of cancer-specific E3 ligase ligands for targeted protein degradation
Authors: Wang Zhaofu , Zhang Lynette , Chang Zhen , Zhao Mengxi , Li Guobin , Qin Fang , Ma Shichao , Li Yunfeng , Zhang Zhongguo , Wang Xinghao , Kong Ying , Lou Shidi , Chen Sheng , Liu Lanzhen , Xu Shuaijie , Zhang Hailong
Published: April 2023

F-box/WD repeat-containing protein 7 (FBXW7) is the substrate recognition component of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex which mediates the ubiquitination and subsequent proteasomal degradation of target proteins. FBXW7 recognizes and binds phosphor-degrons within target proteins and capture them to SCF complex for ubiquitination. Know FBXW7 substrates include cyclin-E, DISC1, JUN, MYC, NFE2L1, NOTCH2, MCL1, RICTOR, and NOTCH1 released notch intracellular domain (NICD). FBXW7 is frequently mutated in cancer, results in loss of substrate recognition and binding. R465C is a one of the major FBXW7 mutations and is found in over 70,000 cancer patients worldwide. From a high-throughput screen, we discovered compounds that selectively bind to FBXW7 R465C mutant. PROTACs against various protein targets have been made with these mutant specific ligands. Some of these PROTACs could induce targeted protein degradation in cells carrying FBXW7 R465C mutant but not wild-type FBXW7. Targeted protein degradation with these PROTACs could represent a novel therapeutic approach against FBXW7 R465C mutant cancer.

Citation Format: Zhaofu Wang, Lynette Zhang, Zhen Chang, Mengxi Zhao, Guobin Li, Fang Qin, Shichao Ma, Yunfeng Li, Zhongguo Zhang, Xinghao Wang, Ying Kong, Shidi Lou, Sheng Chen, Lanzhen Liu, Shuaijie Xu, Hailong Zhang. Discovery of cancer-specific E3 ligase ligands for targeted protein degradation. [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 4962.