bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 138: NIR fluorescent probes for senescence imaging via tunable in situ labeling
Authors: Chen Zixin , Zhang Yuzhao , Anvar Seyedehalaleh , Wu Chang-hung , Liu Jun , Jiang Joyce Z. , Cui Lina
Published: June 2022

Cellular senescence is a mechanism by which a cell enters stable cell cycle arrest triggered by various types of stress such as telomere shortening, oxidative stress and DNA damage. It is implicated in a number of pathophysiological conditions including age-related diseases, degenerative disorders and cancer. Despite its importance, the current toolbox for studying or monitoring senescence in vivo is limited. We have developed a series of novel near-infrared (NIR) fluorescent probes that can be activated by senescence-associated β-galactosidase (SA-β-Gal) and self-immobilize onto intracellular proteins via in situ labeling. We compared the labeling efficiency of these probes in vitro and in cells and the results shows that by modulating the leaving group, we can improve the labeling efficiency of the probes and enhance their retention at the site of activation. These probes can be used for imaging of senescence developed in live cells, as well as long-term tracking of senescent cells in various animal models.

Citation Format: Zixin Chen, Yuzhao Zhang, Seyedehalaleh Anvar, Chang-hung Wu, Jun Liu, Joyce Z. Jiang, Lina Cui. NIR fluorescent probes for senescence imaging via tunable in situ labeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 138.
Abstract 1382: Comparison of intratumoral T-cell repertoires in DNA mismatch repair proficient and deficient colon adenocarcinoma
Authors: Kim Jin K. , Chen Chin-Tung , Khan Asama , Segal Neil , Stadler Zsofia , Shia Jinru , Balachandran Vinod P.
Published: June 2022

Colon tumors with deficient DNA mismatch repair (dMMR) are infiltrated more densely by T cells than tumors with proficient mismatch repair (pMMR). However, high tumor-infiltrating lymphocytes (TIL) are found in a subset of pMMR tumors and low TIL are seen in a subset of dMMR colon cancer. In this study, we compared T-cell repertoires in 20 pMMR and 27 dMMR colon tumors with high and low TIL counts.

CDR3 regions of the TCR beta chain were sequenced by ImmunoSEQ Assay (Adaptive Biotechnologies, Seattle, WA). The fraction T cells was calculated by normalizing TCR beta template counts to the total DNA. Sample richness was calculated as the number of unique productive rearrangements in a sample after computationally down-sampling to a common number of T cells to control for variation in sample depth. Simpson clonality was computed as a measure of how evenly productive rearrangements are distributed amongst the total number of T cells. Statistical analyses were performed using Prism 8.0 (GraphPad, La Jolla, CA).

The mean TIL counts per high power field (HPF) for TIL-high and TIL-low dMMR tumors were 17.4 (range, 5-37) and 1.8 (range, 0-4). In pMMR tumors, they were 12.8 (range, 6-37) for TIL-high and 0.3 (range, 0-1) for TIL-low groups. The number of T cells measured by TCR sequencing correlated positively with the TIL count (Spearman rho = 0.391; P = 0.007). When comparing by MMR status, we found that dMMR tumors exhibited a trend towards higher Simpson clonality index (P = 0.09), with significantly lower repertoire richness compared to pMMR tumors (P = 0.030). Within dMMR tumors, TIL-high dMMR tumors had higher density (P = 0.017), higher clonality (P = 0.003), and a less rich repertoire (P = 0.003) compared to TIL-low dMMR tumors. Within pMMR tumors, while the T cell density was higher in the TIL-high tumors, we did not observe differences in T cell clonality or repertoire richness between TIL-high vs TIL-low pMMR tumors.

Our study found that T cells in dMMR tumors are more clonal and their repertoire is less rich compared with T cells in pMMR tumors. In the dMMR group, T cells in TIL-high tumors were more clonal and their repertoire was less rich compared with T cells in TIL-low tumors, but in the pMMR group, T-cell diversity in TIL-high tumors was comparable to T-cell diversity in TIL-low tumors. These findings suggest that T cells clonally expand in dMMR tumors, possibly in response to MMR deficiency-induced tumor neoantigens.

Citation Format: Jin K. Kim, Chin-Tung Chen, Asama Khan, Neil Segal, Zsofia Stadler, Jinru Shia, Vinod P. Balachandran. Comparison of intratumoral T-cell repertoires in DNA mismatch repair proficient and deficient colon adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1382.
Abstract 1392: Development of a human T cell backbone flow cytometry panel enabling flexibility in reagent choice while minimizing panel design challenges
Authors: Corselli Mirko , Sisouvanthong Chad , Kara Nihan , Widmann Stephanie
Published: June 2022

Advancements in flow cytometry have led to a comprehensive characterization of T cells. While many markers can be used for a detailed immunophenotypic or functional analysis, design of a large multicolor flow cytometry panel is still hampered by the selection of optimal fluorochrome combinations. For example, expansion of existing flow cytometry panels with new markers of interest can result is suboptimal resolution and, in some cases, the need to design a new panel, impacting cost and increasing time to insight. To minimize these challenges and to provide increased flexibility, we have developed a human T cell backbone panel strategically designed to be complemented with 5-8 drop-in markers of choice, depending on instrument configuration. The backbone panel contains five T cell markers (CD3, CD4, CD8, CD45RA, CCR7) conventionally used to identify different maturational states of CD4+ and CD8+ T cells (naïve, central memory, effector memory, effector memory RA). We will show how this backbone panel meets four fundamental requirements: i) clear resolution of major T cell subsets; ii) the fluorochromes used in the backbone panel have minimal resolution impact on the detectors allocated for drop-ins; iii) the fluorochromes assigned to drop-ins have minimal impact on the resolution of the backbone panel; iv) the fluorochromes assigned to drop-ins do not impact each other. We will provide examples of different drop-in combinations for the study of T cell and regulatory T cell biology. Performance of the backbone panel on different instruments will be shown to demonstrate assay consistency across platforms. The compatibility of the backbone panel with intracellular cytokine stain and transcription factor analysis will be further demonstrated. This pre-optimized backbone panel represents a unique tool for researchers who need to study different aspects of T cell biology and want to easily increase their assay capabilities, while minimizing panel design challenges and impact to population resolution. For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Citation Format: Mirko Corselli, Chad Sisouvanthong, Nihan Kara, Stephanie Widmann. Development of a human T cell backbone flow cytometry panel enabling flexibility in reagent choice while minimizing panel design challenges [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1392.
Abstract 1414: Assessment of the Cancer Testis antigen AKAP4, via tumor expression analysis and IHC, as a potential vaccine and immunotherapy target for colorectal cancer
Authors: Herath Mudiyanselage Chandana S. K. , Mitra Neil , Caballero Otavia L. , Gamage Dasuni N. , Yan Xiaohong , Cekic Vesna , Martz Joseph , Whelan Richard L.
Published: June 2022

Introduction: Cancer-Testis antigens (CTA’s) are normally expressed in germline tissue, however, aberrant expression occurs in some cancers. Restricted CTA expression in normal tissues make CTA’s potential vaccine targets. CTA’s have been associated with tumor cell proliferation and invasion. The CTA A-kinase anchor proteins 4 (AKAP 4) plays a role in cancer development and progression. The expression pattern of AKAP4 gene in CRC is not well characterized. This study’s aim was to assess AKAP4 expression in CRC and to assess this CTA’s potential as a vaccine target.

Methods: CRC patients (pts) undergoing surgery who donated tissue to an IRB approved tissue/data bank comprise the study group. Demographic and pathologic data were assessed. Tissues were OCT embedded and stored at -800C. Total purified RNA was isolated from tissue samples and cDNA synthesized. AKAP4 expression was analyzed by quantitative PCR (QPCR) using EXPRESS qPCR universal Supermix and Taqman assay. Comparative quantitative analysis was performed based on the delta-delta Ct method with GAPDH as internal control. Tumor and testis AKAP4 expression levels were determined; tumors with levels 0.1% or more than the testis was considered positive. Immunohistochemistry (IHC) was performed on a subset of tumor and normal tissue. The impact of tumor location and cancer stage on AKAP4 expression was determined and assessed (Wilcoxon signed rank test).

Results: 70 paired CRC and normal tissue specimens (35 M/36 F, age 67.8±14) were studied (86% colon,14% rectal; cancer stage: 1, 24%; 2,37%; 3, 31.%;stg 4, 7%). The percent of pts with a relative Malignant to Normal tissue AKAP4 expression ratio (MN) over 1 was 17%; the percent with both MN ratio over 1 and expression levels above 0.1% of testis levels was 16%. IHC demonstrated AKAP4 in a subset of CRC tumor samples. No significant difference was noted between colon vs rectal, or cancer stage groups. No expression was found in 21 normal samples.

Discussion: In a tumor subset the relative expression of AKAP4 was above that of normal colon and more than 0.1% of testis expression. IHC study demonstrated the antigen in CRC tumors. A larger study is needed determine if AKAP4 expression correlates with T, N, or final tumor stage. AKAP4 holds some promise as a vaccine target.

Citation Format: Chandana S. K. Herath Mudiyanselage, Neil Mitra, Otavia L. Caballero, Dasuni N. Gamage, Xiaohong Yan, Vesna Cekic, Joseph Martz, Richard L. Whelan. Assessment of the Cancer Testis antigen AKAP4, via tumor expression analysis and IHC, as a potential vaccine and immunotherapy target for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1414.
Abstract 1429: DDT induces intergenerational epigenetic inheritance of cancer predisposition in a mouse model
Authors: Da Cruz Raquel Santana , Dominguez Odalys , Nasir Apsra , Gonsiewski Alexandra K. , Cruz Maria Idalia , Jin Lu , McCoy Matthew , de Assis Sonia
Published: June 2022

DNA sequence accounts for the majority of disease heritability, including cancer. Yet, not all familial cancer cases can be explained by genetic factors. There is mounting evidence that environmentally-induced epigenetic inheritance occurs and that the progeny’s traits can be shaped by parental environmental experiences. In humans, epidemiologic studies have implicated endocrine disruptors and environmental toxicants such as the pesticide DDT in intergenerational cancer development, including breast and childhood tumors. Using a mouse model, we investigated whether paternal DDT exposure may result in an intergenerational increase in breast cancer development. We also investigated the effects of DDT on the paternal germline and the mechanisms underlying intergenerational transmission. Male mice (c57bl/6) were exposed to DDT or to a control-vehicle (CO) solution and used for sperm collection or mating with unexposed females to produce the DDT or CO daughters. In another experiment, normal mouse embryos (zygote stage) were injected with RNA purified from sperm of DDT or CO males or miRNA mimic. Resulting zygotes were implanted into surrogate mothers to produce offspring. DDT daughters or RNA injected females were used to study breast cancer development (carcinogen-induced or orthotopic models). We found that the female progeny of males exposed to DDT in the pre-conception period have higher susceptibility of developing aggressive tumors in two different mouse models of breast cancer. Sperm of DDT-exposed males exhibited distinct patterns of small non-coding RNAs, with an increase in miRNAs and a specific surge in miRNA-10b levels. Remarkably, embryonic injection of the entire sperm RNA load of DDT-exposed males or synthetic miRNA-10b recapitulated the tumor phenotypes observed in DDT daughters. We also found that E3.5 embryos injected with miR-10b have altered transcriptional profile compared to controls. Our findings reveal a causal role for sperm RNAs in the inheritance of cancer predisposition and could explain some of the “missing heritability” of breast and other malignancies, if confirmed in humans.

Citation Format: Raquel Santana Da Cruz, Odalys Dominguez, Apsra Nasir, Alexandra K. Gonsiewski, Maria Idalia Cruz, Lu Jin, Matthew McCoy, Sonia de Assis. DDT induces intergenerational epigenetic inheritance of cancer predisposition in a mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1429.
Abstract 1474: Genome edited colorectal cancer organoid models reveal distinct microRNA activity patterns across different mutation profiles
Authors: Villanueva Jonathan W. , Kwong Lawrence , Han Teng , Martinez Salvador Alonso , Pan Fong Cheng , Shanahan Michael T. , Kanke Matt , Chen Shuibing , Dow Lukas E. , Danko Charles G. , Sethupathy Praveen
Published: June 2022

Colorectal cancer (CRC) is the third most common cancer and second leading cause of cancer-related deaths worldwide. DNA mutations drive colon tumor formation by disrupting gene regulatory mechanisms that facilitate cell survival and growth. Distinct combinations of mutations can result in unique changes to these gene regulatory mechanisms leading to variability in responses to available therapeutics. MicroRNAs (miRNAs) are important regulators of gene expression in CRC and it is known that their expression can be altered by oncogenic mutations. However, it is unknown how distinct combinations of CRC-risk mutations differentially affect the regulation and expression of miRNAs. We hypothesize that some miRNAs will exhibit strong genotype-dependent changes in CRC whereas others will be altered irrespective of genotype. Utilizing sequencing data from The Cancer Genome Atlas (TCGA) and cutting-edge intestinal organoid (enteroid) models with different combinations of oncogenic mutations that commonly occur in CRC, we identified several distinct patterns of genotype-dependent miRNA expression. We also found a suite of miRNAs that are up-regulated in a mutation-independent manner. Among these, we demonstrated that miR-24-3p is a candidate master regulator of genes that are down-regulated in all mutational-contexts. RT-qPCR for miR-24-3p in genetically modified human colonoids with different combinations of oncogenic mutations further supports that miR-24-3p is elevated across various mutational contexts. Functional studies in the HCT116 cell line revealed that miR-24-3p controls the number of metabolically active, viable cells by regulating apoptosis but not proliferation. Moreover, follow-up ex vivo studies in mouse enteroids confirmed that miR-24 controls intestinal cell survival. To identify candidate gene targets that mediate the function of miR-24-3p, I performed an RNA-sequencing (RNA-seq) analysis and identified HMOX1 and PRSS8 as top candidate miR-24-3p targets. These findings suggest that miR-24-3p is a genotype-independent regulator of tumor cell apoptosis in CRC and merits further investigation. Overall, our findings provide a novel perspective on approaches for precision medicine in CRC.

Citation Format: Jonathan W. Villanueva, Lawrence Kwong, Teng Han, Salvador Alonso Martinez, Fong Cheng Pan, Michael T. Shanahan, Matt Kanke, Shuibing Chen, Lukas E. Dow, Charles G. Danko, Praveen Sethupathy. Genome edited colorectal cancer organoid models reveal distinct microRNA activity patterns across different mutation profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1474.
Abstract 1484: Emerging microRNAs as biomarkers for colorectal cancer diagnosis: A meta-analysis
Authors: Fathi Sofia , Guessous Fadila , Karkouri Mehdi
Published: June 2022

Background: Colorectal cancer (CRC) is the second leading cause of cancer deaths worldwide. The current method of clinical screening is mainly based on colonoscopy. However, many CRC patients are unwilling to undergo colonoscopy because of the high procedure cost and the discomfort during examination. Therefore, it appears necessary to find novel and non-invasive biomarkers that would improve CRC early detection. MicroRNAs (miRNAs) are a class of non-coding RNAs with critical regulatory roles in fundamental cellular processes, such as proliferation, migration, and apoptosis. They can act as potential oncogenes or tumor suppressors in cancer development. Numerous studies have shown significant differences in miRNAs expression in serum or plasma of CRC patients and healthy subjects. Thus, the potential use of circulating miRNAs as diagnostic biomarkers in CRC seems to be a fast and relevant alternative to traditional and invasive methods. Here, we reviewed selected studies which identified dysregulated circulating miRNAs in CRC and evaluated their diagnostic value.

Methods: This meta-analysis study was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The articles were searched in online databases such as PubMed, Google Scholar to identify all eligible studies that evaluated the diagnostic value of miRNAs for CRC. Then we explored the diagnostic accuracy of each miRNA using the random-effects model to estimate the pooled sensitivity (Se), specificity (Sp), Positive likelihood ratio (PLR) and Negative likelihood ratio (NLR) and the area under the curve (AUC), along with their corresponding 95% confidence intervals (CIs). The statistical analyses were performed using the Review Manager (Revman) 5.4 software and the Meta Disc software.

Results: Thirteen studies involving five microRNAs were included in our analysis. Our results suggested that serum miR-29a levels showed great potential as a high diagnostic biomarker for CRC. The overall pooled sensitivity, specificity, area under the curve (AUC), PLR and NLR of circulating miR-29a were 69.75%, 88.25%, 0.8351, 5.93 and 0.342 respectively.

Conclusion: miRNAs have become increasingly recognized molecular tools in CRC diagnosis. According to our results, circulating miR-29a level has potential value for early detection and might be used as a novel diagnostic biomarker for CRC in combination with other biomarkers.

Citation Format: Sofia Fathi, Fadila Guessous, Mehdi Karkouri. Emerging microRNAs as biomarkers for colorectal cancer diagnosis: A meta-analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1484.
Abstract 1486: MicroRNA (miR)199a-5p targets JUN-B to decrease cell proliferation in esophageal cancer cells
Authors: Phatak Pornima , Tulapurkar Mohan E. , Donahue James M.
Published: June 2022

Objectives: MiR-199a-5p has been shown to be downregulated in multiple malignancies and function as a tumor suppressor. We have previously demonstrated that miR-199a-5p is markedly downregulated in esophageal cancer cell lines compared to esophageal epithelial cells. In several miR-target sequence analysis programs, miR-199a-5p is predicted to bind JUN-B mRNA with high affinity. JUN-B is an important component of the AP1 transcription factor. The objective of this study was to determine expression of JUN-B in esophageal cancer cells as well as to investigate the interaction between miR-199a-5p and JUNB in these cells and to characterize the functional implications of this interaction.

Methods: Studies were performed in human esophageal epithelial (hESO) cells and in TE7, SK-GT-4 and OE21 human esophageal cancer cells. Expression of miR-199a-5p and JUNB mRNA in these cell lines was measured by real-time PCR. JUNB protein expression levels were examined by Western blot. Function of miR-199a-5p was tested through its overexpression and silencing. Cellular proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Results: Levels of miR-199a-5p in TE7, SK-GT-4, and OE21 esophageal cancer cells are markedly reduced compared to hESO cells. JUN-B mRNA and protein expression are correspondingly elevated in these cells compared to hESO cells. JUN-B mRNA and protein expression levels were decreased following miR-199a-5p overexpression in TE7, SK-GT-4 and OE21 cells. In reciprocal experiments, silencing miR-199a-5p in hESO cells resulted in increased JUN-B protein and mRNA levels. Forced expression of miR-199a-5p in TE7, SK-GT-4 and OE21 cells led to a marked decrease in cellular proliferation.

Conclusions: MiR-199a-5p expression is significantly reduced in TE7, SK-GT-4, and OE21 esophageal cancer cells relative to hESO cells. Forced expression of miR-199a-5p results in significantly decreased JUN-B mRNA and protein expression in these cells, leading to decreased cellular proliferation. These results add our understanding of the roles by which miR-199a-5p functions as a tumor suppressor in esophageal cancer cells. Further studies will investigate the association of miR-199a-5p with JUN-B mRNA using biotinylated RNA pull-down and luciferase reporter assays, in addition to determining the effect of Jun-B downregulation on AP1 promoter activity.

Citation Format: Pornima Phatak, Mohan E. Tulapurkar, James M. Donahue. MicroRNA (miR)199a-5p targets JUN-B to decrease cell proliferation in esophageal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1486.
Abstract 1490: Development of a 5-FU modified miR-129 mimic as a novel therapeutic for non-small cell lung cancer
Authors: Hwang Ga-Ram , Yuen John G. , Farley Hannah , Ju Jingfang
Published: June 2022

Lung cancer is the leading cancer type of cancer-related deaths in the U.S., with non-small cell lung cancer (NSCLC) accounting for a majority of cases. Despite innovations in cancer therapeutics, due to resistance to these therapeutics, the 5-year survival rate for patients with metastatic lung cancer has remained poor. As a result, there is an urgent need to develop new and effective therapeutics, including the study of RNA-based therapeutics. Previous studies from our laboratory have shown that 5-fluorouracil (5-FU) modified tumor suppressor miRNAs significantly enhance the therapeutic efficacy, stability, and deliverability of miRNAs in colorectal cancer (CRC) and pancreatic cancer. In this study, we have characterized the therapeutic potential of miR-129 modified with 5-FU (5-FU-miR-129) in non-small cell lung cancer in vitro via Annexin V staining, cell cycle analysis, and Western Blot. 5-FU-miR-129’s therapeutic potential was also assessed in vivo via IVIS imaging of NSCLC xenografts. We have demonstrated that 5-FU-miR-129 can induce apoptosis (7.2-fold in A549 and 2.9-fold in Calu-1) and cell cycle arrest (12.7-fold change in G1/S phase ratio in A549) in NSCLC in vitro. Furthermore, 5-FU-miR-129 has been found to retain target specificity by downregulating miR-129 targets BCL2 and HMGB1 in a cell line-dependent manner. As was observed in previous studies, we have also demonstrated the ability of 5-FU-miR-129 to be delivered in cancer cells without the use of a transfection vehicle in vitro. Finally, we were also able to demonstrate the therapeutic potential of 5-FU-miR-129 in inhibiting NSCLC tumor growth in vivo, as seen by a significantly decreased change in tumor growth by 37 days post-treatment in the 5-FU-miR-129 treated group compared to the negative control group (79.8% reduction in tumor growth). In addition, we observed a significant increase in survival probability and a reduced hazard ratio in mice treated5-FU-miR-129 when compared to the negative control group. Furthermore, we did not observe any visible side effects (hair loss) of mice treated with 5-FU-miR-129 compared to chemotherapeutic drugs 5-FU and gemcitabine (Gem). 5-FU-miR-129’s therapeutic potential in NSCLC further demonstrates the potential for modified miRNAs as a platform technology in cancer therapeutics.

Citation Format: Ga-Ram Hwang, John G. Yuen, Hannah Farley, Jingfang Ju. Development of a 5-FU modified miR-129 mimic as a novel therapeutic for non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1490.
Abstract 1495: Bioengineered RNAi molecules for cancer research and experimental therapy
Authors: Yu Ai-Ming
Published: June 2022

RNAs have emerged as a novel group of medications, with distinct chemistry and pharmacology from conventional small molecules and proteins or antibodies. Indeed, some RNAi products (e.g., siRNAs) have been approved for clinical practice by the United State Food and Drug Administration while many others, including genome-derived miRNAs, are under active investigations. However, current research and development relies predominantly on the use of chemo-engineered RNA mimics made in vitro that are different from natural RNA molecules produced and folded in vivo. This is also in sharp contrast to protein research and therapy that primarily uses bioengineered or recombinant proteins produced and folded in vivo, instead of synthetic polypeptides or proteins made in vitro. Therefore, we have devoted large efforts to producing true biologic RNA molecules to better recapitulate the properties of natural RNA molecules for research and drug development. Herein I will present our one-of-a-kind RNA bioengineering technology that achieves high-yield, large-scale and cost-effective, in vivo fermentation production of biologic RNAs (BioRNAs) consisting of target RNAi molecules (e.g., miRNAs or siRNAs). Examples will be presented to demonstrate the precise release of payload RNAi molecules from BioRNAs in human cells and subsequently, modulate target gene expression and various cancer cellular processes. In addition, biologic RNAi prodrugs are effective to control tumor growth and metastasis in clinically relevant xenograft mouse models. Our results demonstrate that bioengineered RNA molecules are a novel class of agents for cancer research and drug development. Acknowledgements: This study was supported by grants R01CA225958, R01CA253230 and R35GM140835 from NIH.

Citation Format: Ai-Ming Yu. Bioengineered RNAi molecules for cancer research and experimental therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1495.
Abstract 1504: Mouse model and human patient data suggest critical roles for Pten and p53 in suppressing POLE mutant tumor development
Authors: Sun Meijuan J. , Park Vivian S. , Frey Wesley D. , Williams Leonard G. , Hodel Karl P. , Strauss Juliet D. , Wellens Sydney J. , Jackson James G. , Pursell Zachary F.
Published: June 2022

Mutations in the exonuclease domain of POLE are associated with tumors harboring very high mutation burdens. The mechanisms linking this significant mutation accumulation and tumor development remain poorly understood. Pole+/P286R;Trp53+/- mice showed accelerated cancer mortality compared to Pole+/P286R;Trp53+/+ mice. Cells from Pole+/P286R mice showed increased p53 activation, and subsequent loss of p53 permitted rapid growth, implicating canonical p53 loss of heterozygosity in POLE mutant tumor growth. Somewhat surprisingly, however, p53 status had no effect on tumor mutation burden or single base substitution signatures in POLE mutant tumors from mice or humans. Pten has important roles in maintaining genome stability. We find that PTEN mutations are highly enriched in human POLE mutant tumors, including many in POLE signature contexts. One such signature mutation, PTEN-F341V, was previously shown in a mouse model to specifically decrease nuclear Pten and lead to increased DNA damage. We found tumors in Pole+/P286R mice that spontaneously acquired PtenF341V mutations and were associated with significantly reduced nuclear Pten and elevated DNA damage. Further analysis in cells suggests the presence of the mutant polymerase triggers DNA damage in specific contexts. Taken together, our analysis supports the idea that POLE-mediated hypermutagenesis is necessary, but not entirely sufficient, for tumorigenesis. Disabling surveillance of nuclear DNA damage is a likely sufficient factor.

Citation Format: Meijuan J. Sun, Vivian S. Park, Wesley D. Frey, Leonard G. Williams, Karl P. Hodel, Juliet D. Strauss, Sydney J. Wellens, James G. Jackson, Zachary F. Pursell. Mouse model and human patient data suggest critical roles for Pten and p53 in suppressing POLE mutant tumor development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1504.
Abstract 1510: Exposure to butyrate metabolite mediates genomic instability in oropharyngeal keratinocytes
Authors: McGhee Eva , Li Mengtao , Lin Yi-Ling , Su-Velez Brooke , Carr Nefertari , Njubigbo Chinelo , Sadeghi Rohun , Sadegh Matin , Handler Julian , Handler Adin , Long Naomi , Vadgama Jay
Published: June 2022

Human Papillomavirus (HPV), a non-enveloped epitheliotropic double stranded DNA virus, is an etiological agent of oral cancer, and can be cleared by host immunity. A small percentage of patients that develop persistent infection with oncogenic HPV show an increased risk of developing an HPV-associated malignancy. Head and neck, oropharyngeal cancer is related to the persistent infection by high-risk HPV type 16, E6/E7 oncoproteins. The E6/E7 oncoproteins significantly contribute to the carcinogenic genomic instability effect of high-risk HPV through the degradation of two proteins, p53 and pRB, respectively. The human oropharynx often harbors anaerobic bacteria that produce a variety of byproducts, including butyrate. Butyrate is a modulator of induced genomic instability, influencing the development of HPV oral cancer when implanted in a syngeneic mouse model. In this study, we investigate butyrate co-treatment with HPV E6/E7 in vitro. This treatment increased cellular proliferation, DNA double strand breaks, and genetic instability processes associated with apoptosis regulation. These data indicate that in connection with butyrate, HPV E6/E7 oncoproteins can promote increased upregulation of apoptosis, disrupt cell cycle regulation, and decrease DNA double strand break repair. Epigenetics and chromosomal instability events have been noted, specifically, the deletion of the distal region of chromosome 11q22-23 in E6/E7 transfected keratinocytes. These results indicate that HPV-16, E6/E7 oncoproteins plus butyrate treatment can influence genomic changes in mouse keratinocytes, which may further promote oral carcinogenesis.

Citation Format: Eva McGhee, Mengtao Li, Yi-Ling Lin, Brooke Su-Velez, Nefertari Carr, Chinelo Njubigbo, Rohun Sadeghi, Matin Sadegh, Julian Handler, Adin Handler, Naomi Long, Jay Vadgama. Exposure to butyrate metabolite mediates genomic instability in oropharyngeal keratinocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1510.
Abstract 1528: Molecular mechanisms of TSLP as a therapy for CRLF2 B-Cell acute lymphoblastic leukemia
Authors: Alkashgari Hossam , Stoian Cornelia , Ruiz-Jimenez Caleb , Coats Jacqueline , Casiano Carlos A. , Dovat Sinisa , Payne Kimberly J.
Published: June 2022

B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of leukemia in children. B-ALL characterized by cytokine receptor-like factor 2 (CRLF2) overexpression (CRLF2 B-ALL) has a survival rate of <30% and is the highest risk sub-group of B-ALL in both adults and children. CRLF2 is a receptor component for the cytokine thymic stromal lymphopoietin (TSLP). TSLP plays a role in the survival and proliferation of B-cell precursors, thus explaining the oncogenic role of increased CRLF2 signaling in CRLF2 B-ALL. To our surprise, we found that high-levels of TSLP eliminated leukemia cells in patient-derived xenograft (PDX) models of CRLF2 B-ALL. CRLF2 and IL-7 receptor-alpha (IL-7Ra) form the heterodimer type-I cytokine receptor for TSLP cytokine. Binding of TSLP to its CRLF2 receptor complex induces JAK-STAT5 and PI3K-AKT pathway signals. TSLP shares the IL-7Ra with Interleukin 7 (IL-7) which has a heterodimer receptor consisting of IL-7Ra and the common gamma chain. High-levels of IL-7 (50 ng/ml) have been shown to induce IL-7Ra internalization and degradation in T-cells. We hypothesize that high-level TSLP induces internalization and degradation of IL-7Ra leading to CRLF2 signal inhibition, death of CRLF2 B-ALL cells and the anti-leukemia effects that we have observed in PDX mice. To test this hypothesis, we treated CRLF2 B-ALL cell lines with different TSLP concentrations to observe the effect of TSLP on its receptor and CRLF2 signaling. Flow cytometry data showed that continuous or a pulse of high-dose TSLP induced a loss of surface IL-7Ra expression for up to 24 hours. Phosphorylation assays showed that cells cultured with high-dose TSLP were unresponsive to subsequent TSLP-induced phosphorylation events (pSTAT5 and pRPS6), indicating CRLF2 signal inhibition. In conclusion, high-dose TSLP induces loss of (IL-7Ra) and inhibition of CRLF2 signaling. These results suggest that TSLP exerts its anti-leukemia effects by shutting down CRLF2-mediated signals possibly via the loss of the IL-7Ra receptor component.

Citation Format: Hossam Alkashgari, Cornelia Stoian, Caleb Ruiz-Jimenez, Jacqueline Coats, Carlos A. Casiano, Sinisa Dovat, Kimberly J. Payne. Molecular mechanisms of TSLP as a therapy for CRLF2 B-Cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1528.
Abstract 1556: Long non-coding RNA WNT5A-AS1 shows sex-dimorphic differences in survival for males with glioblastoma
Authors: Miller Aunay , Wang Junwen , Loftus Joseph , Tran Nhan L.
Published: June 2022

Glioblastoma (GBM) is the most common and aggressive primary brain tumor with a median survival time of 12-15 months and 5-year survival rate of 5%. Despite the application of advanced genetics and biological breakthroughs in GBM, the outcome for GBM remains dismal. Previous genome-wide association studies (GWAS) for GBM shows that the incidence rate for GBM in males is 1.6 times higher in men than women, with men displaying overall worse outcomes. In-vivo studies in gender-matched isogenic murine astrocytic cell lines show that the rate of astrocytic transformation is higher in males than in females, corroborating the GWAS findings in human GBM. Thus, understanding how sex effects GBM biology may provide insight into the mechanism of gliomagenesis and response to therapy. To explore the molecular mechanisms by which transformation of GBM increases in males, we used DESeq2 to assess differentially expressed genes between male and female patients. We found that a long non-coding RNA WNT5A-AS1 is overexpressed in males [log2(fold change) of 1.40; FDR = 0.036], regardless of cofounding factors such as IDH1 status, age, and race. Next, we examined if expression of WNT5A-AS1 is correlated with survival within male patients versus female patients in the TCGA-GBM dataset. In males, WNT5A-AS1’s expression is significantly correlated with patient overall survival (P = 0.042, N=109), but not in female (P = 0.585, N=60). Male patients with high expression of WNT5A-AS1 display poor survival outcomes compared to males with low expression. WNT5A-AS1 is located in the upstream, promoter region of WNT5A. Q-PCR quantification of WNT5A expression increases in male GBM specimens as compared to GBM specimens from females. The role that WNT5A-AS1 plays in regulating WNT5A expression is unclear to date. Interestingly, WNT5A expression shows a positive correlation with patient survival in males, with high expression displaying better survival outcomes (p=0.0272). We have data obtained from a subset of patient samples showing an inverse relationship between WNT5A and WNT5A-AS1 expression. This suggests that there may be an inverse correlation between WNT5A-AS1 and WNT5A expression, which elucidates that WNT5A-AS1 may be regulating WNT5A expression. Current ongoing studies will evaluate the role of WNT5A-AS1/WNT5A in sex-differences of GBM progression.

Citation Format: Aunay Miller, Junwen Wang, Joseph Loftus, Nhan L. Tran. Long non-coding RNA WNT5A-AS1 shows sex-dimorphic differences in survival for males with glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1556.
Abstract 1567: Increased extracellular matrix stiffness induces hypersecretion of chemoresistance-promoting cancer associated fibroblast-derived exosomes in pancreatic cancer
Authors: Xiao Weikun , Eun Chae-Young , Zhang Xinyu , DeKalb Charlene , Pahlavan Mahsa , Mahgoub Bayan , Knaneh Hanaa , Sohrabi Alireza , Seidlits Stephanie K. , Hill Reginald
Published: June 2022

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer with few effective treatments. Despite recent advances in in other cancers, the five-year survival rate of PDAC is still only 10%, with most patients succumbing to their disease within the first year. One of the main factors responsible for this poor outcome is the development of chemoresistance in nearly all clinical cases. While the intrinsic factors that facilitate chemoresistance in the tumor cells have been studied extensively, fewer studies have elucidated how the complex and unique microenvironment surrounding the tumor affects therapeutic responses. The abundant stromal cells and the stiff desmoplastic microenvironment constitute more than 90% of the primary tumor bulk. However, there is a lack of high-throughput, easily tunable models to recapitulate this complex microenvironment, in order to identify a critical cell-extrinsic factors that could drive acquired chemoresistance in PDAC cells. To address this issue, we have developed a Matrigel-based, orthogonally tunable 3-dimensional (3D) culture system to co-culture mouse derived PDAC organoids and host-matching cancer-associated fibroblasts (CAFs). Using this system, we found that matrix-activated CAFs readily remodel the desmoplastic matrix through lysol-oxidase dependent crosslinking. Moreover, our novel system demonstrates how collagen-I and matrix stiffness affect chemoresistance via the hypersecretion CAF-derived exosomes. Interestingly, our data show that it is CAF-derived exosomes, not the organoid-derived exosomes, that are most important in regulation of proliferation and chemoresistance. Lastly, we identified key cell surface integrins for collagen-I as therapeutic targets to prevent desmoplastic matrix-mediated chemo-resistance in PDAC. Our study provides insights into how matrix composition and stiffness affect therapeutic outcome in PDAC. Moving forward, therapies aimed at interrupting how PDAC cells and stomal cells sense the matrix microenvironment could be utilized to eventually overcome matrix-mediated chemoresistance in PDAC.

Citation Format: Weikun Xiao, Chae-Young Eun, Xinyu Zhang, Charlene DeKalb, Mahsa Pahlavan, Bayan Mahgoub, Hanaa Knaneh, Alireza Sohrabi, Stephanie K. Seidlits, Reginald Hill. Increased extracellular matrix stiffness induces hypersecretion of chemoresistance-promoting cancer associated fibroblast-derived exosomes in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1567.
Abstract 1574: LSD-germline variant drive oncogenicity
Authors: Yoon Hyundong , Song Seulki , Ha Yeeun , Koh Youngil , Yoon SungSoo
Published: June 2022

Lysosome storage disease (LSD) is a congenital metabolic disorder caused by mutations in genes involved in the function of lysosomes. Defects in the genes encoding hydrolases and transporters and enzyme activators are found in LSD’s, leading to the accumulation of macromolecules in the late endocytic system. And this disruption of lysosomal homeostasis increases the induction of endoplasmic reticulum and oxidative stress, proliferative signal transduction, extracellular matrix degradation, Integrin-β4-mediated cell migration and invasion, and consequently can promote the development of oncogenic cells and malignant tumors. It is known that the function of lysosomes is very important in cancer diseases. Recently, through a preliminary study, we found that the incidence of myeloproliferative neoplasm (MPN) increases statistically significantly when the LSD putative pathogenic germline variant (PPGV) is present. Based on this, we performed single-cell RNA (scRNA) sequencing using MPN patient samples of both LSD PPGV carrier and non-carrier. We tried to determine how the LSD PPGV affects MPN cancer progression at the single-cell level. To confirm functional differences between the two groups, DEG, pathway enrichment, LR-pair interaction, and transcription enrichment analyses were performed. In the analysis, immune cells were composed of T-cell, NK-cell, and Monocyte (in the order of frequency). It was confirmed that Lysosome-related (ER, Golgi, etc.) functions were upregulated in the carrier group compared to the non-carrier group across cell types. In addition, in the ligand-receptor (LR)-pair interaction analysis targeting Immune cells, it was possible to find a statistically significant difference (average log2 fold change>0.7 and p-value<0.05) candidate LR-pair (IL6-IL6R*IL6ST) that upregulates in the carrier group compared to the non-carrier group. The candidate LR-pair found by our team has been previously known as a cytokine-related gene that plays an inflammatory or fibrosis-related role in MPN and affects cancer progression. Also, in the Transcription Factor Enrichment analysis conducted together, it was confirmed that the statistically significant (NES>2 and FDR<0.01) candidate Transcription Factor (STAT1), which operates dependent on the candidate LR-pair, is upregulated. In conclusion, we found the possibility that genes and functions related to cancer progression can be up-regulated by LSD germline variants. In addition, we plan to conduct additional studies to determine the exact mechanism of contribution to cancer development by the LSD germline variant.

Citation Format: Hyundong Yoon, Seulki Song, Yeeun Ha, Youngil Koh, SungSoo Yoon. LSD-germline variant drive oncogenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1574.
Abstract 1599: Genes subject to changes in heterogeneity can function as regulators of metastasis
Authors: Yang Dongbo , Dann Christopher , Valdespino Andrea , Robinson-Mailman Lydia , Chen Mengjie , Pott Sebastian , Rosner Marsha
Published: June 2022

Tumor cell heterogeneity has been strongly implicated in metastatic progression of solid tumors such as triple-negative breast cancer (TNBC), leading to resistance and recurrence. Raf Kinase Inhibitory Protein (RKIP or PEBP1) effectively suppresses metastasis in TNBC but is lost in many patients. We hypothesize that RKIP works in part by reducing the variability of gene expression in TNBC cells. To test whether RKIP reduces overall transcriptional heterogeneity, we compared the variation in gene expression between individual cells in metastatic (low RKIP) versus non-metastatic (high RKIP) TNBC tumors. Single-cell RNA-sequencing revealed transcriptional heterogeneity was decreased with RKIP overexpression. Many cancer regulatory genes were more homogeneous and up-regulated in the RKIP-expressing cells. Among these enriched and homogenously expressed genes is KMT5C, a histone methyltransferase that facilitates epigenetic transcriptional repression. Cell, mouse, and clinical studies suggest KMT5C expression blocks invasion, extravasation, and colonization of tumor cells, consistent with a role as a novel suppressor of metastasis. These studies reveal the importance of genes involved in heterogeneity as potential regulators of metastatic progression in cancer.

Citation Format: Dongbo Yang, Christopher Dann, Andrea Valdespino, Lydia Robinson-Mailman, Mengjie Chen, Sebastian Pott, Marsha Rosner. Genes subject to changes in heterogeneity can function as regulators of metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1599.
Abstract 171: ERK dynamics are essential for colon tissue patterning
Authors: Pond Kelvin W. , Chakrabarti Jayati , Thorne Curtis A. , Paek Andrew L.
Published: June 2022

Maintaining the correct ratio of cellular niches in space and time allows tissues to perform their complex functions and create patterns. This maintenance of tissue patterning is often dysregulated during cancer development. In order to study tissue patterning, temporal and spatial resolution of communication between single cells is required. To explore how this complex process is controlled, we developed a patient-derived organoid screening platform to study both tissue patterning and kinase pathway dynamics in single cells across hundreds of conditions. Unlike normal and polyp-derived organoids, tumor organoids displayed a lack of patterning reminiscent of in vivo dysplasia. Using this system, we discovered that waves of Erk signaling induced by apoptotic cells are critical for maintaining tissue patterning in human colonic organoids. Using a small molecule-based screen, we discovered novel kinases which limit Erk activation across tissues and help maintain patterning. Perturbation of Erk restricting kinases results in dysfunctional colonic wound repair and re-establishment of tissue patterns in vivo.

Citation Format: Kelvin W. Pond, Jayati Chakrabarti, Curtis A. Thorne, Andrew L. Paek. ERK dynamics are essential for colon tissue patterning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 171.