Important Role Research Articles

Selective genes expression and metabolites transformation drive a robust nitrite accumulation during nitrate reduction under alternating feast-famine condition

Nitrite production via denitrification has been regarded as a key approach for survival of anaerobic ammonium oxidation (anammox) bacteria. Despite the important carbon substrate, little is known about the role of differential genes expression and extracellular metabolite regulation among diverse microbial communities. In this study, a novel alternating feast-famine strategy was proposed and demonstrated to efficiently accumulate nitrite in a low-nitrogen loading rate (NLR) (0.2∼0.8 kg N/m3/d) denitrification system. Highly selective expression of denitrifying genes was revealed as key regulators. Interestingly, in absence of carbon source (ACS) condition, the expression of narG and narI/V genes responsible for reduction of nitrate to nitrite jumped to 2.5 and 5.1 times higher than that in presence of carbon source (PCS) condition with carbon to nitrate ratio of 3.0. This fortunately facilitated a rapid nitrite accumulation once acetate was added, despite a significantly down-regulated narG and narI/narV and up-regulated nirS/nirK. This strategy selected Thauera as the most dominant denitrifier (50.2 %) with the highest contribution to narG and narI/narV genes, responsible for the high nitrite accumulation. Additionally, extracellular xylose, pyruvate, and glucose jointly promoted carbon-central metabolic pathway of key denitrifiers in ACS stage, playing an important role in the process of self-growth and selective enrichment of functional bacteria. The relatively rapid establishment and robust performance obtained in this study shows an engineering-feasible and economically-favorable solution for the regulation of partial denitrification in practical application.

Build Your Own Robot Friend: An Open-Source Learning Module for Accessible and Engaging AI Education

As artificial intelligence (AI) is playing an increasingly important role in our society and global economy, AI education and literacy have become necessary components in college and K-12 education to prepare students for an AI-powered society. However, current AI curricula have not yet been made accessible and engaging enough for students and schools from all socio-economic backgrounds with different educational goals. In this work, we developed an open-source learning module for college and high school students, which allows students to build their own robot companion from the ground up. This open platform can be used to provide hands-on experience and introductory knowledge about various aspects of AI, including robotics, machine learning (ML), software engineering, and mechanical engineering. Because of the social and personal nature of a socially assistive robot companion, this module also puts a special emphasis on human-centered AI, enabling students to develop a better understanding of human-AI interaction and AI ethics through hands-on learning activities. With open-source documentation, assembling manuals and affordable materials, students from different socio-economic backgrounds can personalize their learning experience based on their individual educational goals. To evaluate the student-perceived quality of our module, we conducted a usability testing workshop with 15 college students recruited from a minority-serving institution. Our results indicate that our AI module is effective, easy-to-follow, and engaging, and it increases student interest in studying AI/ML and robotics in the future. We hope that this work will contribute toward accessible and engaging AI education in human-AI interaction for college and high school students.

Theoretical Investigation on Electronic Excited States of the HSO Radical.

HSO radicals play an important role in the photochemical processes in combustion, the atmosphere, and the interstellar medium. In this work, we perform a high-level ab initio study on the electronic excited states of HSO using the internally contracted multireference configuration interaction methods including Davidson correction (icMRCI + Q) in combination with the correlation-consistent basis sets. The molecular geometries, vertical transition energies, oscillator strengths, and electronic configurations of 19 electronic states of HSO are computed. The electronic potential energy curves of HSO along the bond lengths and bond angles are presented. Based on our calculations, the interactions between the electronic states involved in the ultraviolet region and the mechanism of photodissociation are discussed, which will lay a foundation for revealing the dissociation dynamics of gas-phase HSO molecules in outer space and the earth's atmosphere.

Identification of Anoikis-Related Genes in Spinal Cord Injury: Bioinformatics and Experimental Validation.

Spinal cord injury (SCI) is a serious disease without effective therapeutic strategies. To identify the potential treatments for SCI, it is extremely important to explore the underlying mechanism. Current studies demonstrate that anoikis might play an important role in SCI. In this study, we aimed to identify the key anoikis-related genes (ARGs) providing therapeutic targets for SCI. The mRNA expression matrix of GSE45006 was downloaded from the Gene Expression Omnibus (GEO) database, and the ARGs were downloaded from the Molecular Signatures Database (MSigDB database). Then, the potential differentially expressed ARGs were identified. Next, correlation analysis, gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and protein-protein interaction (PPI) analysis were employed for the differentially expressed ARGs. Moreover, miRNA-gene networks were constructed by the hub ARGs. Finally, RNA expression of the top ten hub ARGs was validated in the SCI cell model and rat SCI model. A total of 27 common differentially expressed ARGs were identified at different time points (1, 3, 7, and 14days) following SCI. The GO and KEGG enrichment analysis of these ARGs indicated several enriched terms related to proliferation, cell cycle, and apoptotic process. The PPI results revealed that most of the ARGs interacted with each other. Ten hub ARGs were further screened, and all the 10 genes were validated in the SCI cell model. In the rat model, only seven genes were validated eventually. We identified 27 differentially expressed ARGs of the SCI through bioinformatic analysis. Seven real hub ARGs (CCND1, FN1, IGF1, MYC, STAT3, TGFB1, and TP53) were identified eventually. These results may expand our understanding of SCI and contribute to the exploration of potential SCI targets.

Fracture density reconstruction using direct sampling multiple-point statistics and extreme value theory

The aim of this work is to present a methodology for the reconstruction of missing fracture density within highly fractured intervals, which can represent preferential fluid flow pathways. The lack of record can be very common due to the intense presence of fractures, dissolution processes, or data acquisition issues. The superposition of numerous fractures makes the definition of fracture surfaces impossible, as a consequence, modeling such zones is challenging. In order to address this issue, the usage of direct sampling multiple-point statistics to perform gap filling in well logs is demonstrated as an alternative to other techniques. It reproduces data patterns and provides several models representing uncertainty. The method was tested in intervals from a highly fractured well, by removing previously known fracture density data, and simulating different scenarios with direct sampling. Simulation results are compared to the observed data using cross-validation and continuous rank probability score. The reference scenario training data set consists in one well and two variables: fracture density and fracture occurrence. A sensitivity analysis is carried out considering additional variables, additional wells, different intervals, resampling with extremes, and other gap filling techniques. The auxiliary variable plays an important role in pattern matching, but adding wells and logs increases the complexity of the method without improving pattern retrieval. Best results are obtained applying extreme values theory for stochastic process with the enrichment of the fracture density data at the tail region, followed by resampling of the new values. The enriched data is used for the gap filling resulting in lower continuous rank probability score, and the achievement of extreme fracture density values.

Abstract 5332: ST316, a clinical peptide antagonist of beta-catenin, induces anti-tumor immune responses by multiple mechanisms of action

Abstract Wnt/β-catenin plays several important roles in cancer, including driving oncogenesis via cell proliferation, survival and metabolic reprogramming of cancer cells, and enhancing immune-desertification of the tumor immune microenvironment (TIME). ST316 is a clinical stage cell-penetrating peptide antagonist of the interaction of β-catenin with its co-activator BCL9, selectively impairing a subset of Wnt target genes and demonstrating potent anti-tumor activity against Wnt-driven tumors in vivo. ST316 is currently being evaluated in a Phase 1-2 study (NCT05848739) enrolling patients with selected advanced solid tumors likely to harbor abnormalities of the Wnt/β-catenin signaling pathway. Here we explore the potential of ST316 to activate the TIME and provide data to support combination treatment with anti-PD-1 and anti-TIGIT therapies. Macrophages derived from human peripheral blood mononuclear cells (hPBMCs) were activated by LPS and IFNγ (M1) or IL-4 (M2) in the presence of ST316 or control and were immunophenotyped by expression of CD80 and CD163 to assess M1 and M2 macrophages, respectively. ST316 induced marked repolarization of hPBMC-derived M2 macrophages to the M1 identity in vitro, as shown by increased CD80 and decreased CD163 staining. Increasing concentration of ST316 in co-cultures of M2 cells with CD8+ T cells induced up to a three-fold increase in IFN-γ expressing cells relative to controls. Additionally, ST316 exposure increased CD155/PVR expression on 4T1 Triple Negative Breast Cancer (TNBC) cells, but not MV411 TNBC that are not Wnt-dependent. When combined with anti-TIGIT treatment, ST316 led to increased T cell activation in an ex vivo assay where 4T1 cells were co-incubated with syngeneic splenic CD8+ cells and frequency of IFN-γ producing cells was assessed. In in vivo experiments, Balb/C mice bearing syngeneic 4T1 TNBC orthotopic tumors were treated with vehicle, ST316, anti-PD-1 or combination. Sub-pharmacologic ST316 enhanced anti-PD-1 suppression of 4T1 tumor growth and induced a substantial decrease of the M2 marker CD209 (DC-SIGN) in the Tumor Associated Macrophage (TAMs). These data support two novel mechanisms of action for the β-catenin antagonist peptide ST316. First, ST316 exposure results in macrophage repolarization towards an immune-active M1 program, both in vitro and in vivo, and increases T-cell activation in co-culture assays. Second, ST316 induced CD155/PVR upregulation and T-cell activation in the presence of anti-TIGIT antibody. Collectively these results suggest a novel immune-modulatory role for ST316 in the TIME and provide rational for combination therapy with checkpoint inhibitors. Citation Format: Claudio Scuoppo, Karen Mendelson, Ricardo Ramirez, Mark Koester, Julia Diehl, Erin Gallagher, Siok Leong, Jerel Gonzales, Zach Mattes, Lila Ghamsari, Gene Merutka, Barry J. Kappel, Abi Vainstein-Haras, Jim A. Rotolo. ST316, a clinical peptide antagonist of beta-catenin, induces anti-tumor immune responses by multiple mechanisms of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5332.

Abstract 5770: Inhibition of KRAS G12D mutant with small molecules

Abstract Background: RAS mutations are occurring at a high rate in colorectal cancer, lung cancer, and pancreatic cancer. RAS is family of small GTPase proteins with important roles in cell growth, differentiation, proliferation, and survival. There are three types of RAS genes: HRAS, KRAS, and NRAS. The The RAS gene is the most commonly found oncogene in human cancer. KRAS exhibits a highest mutation rate compared to those of HRAS and NRAS in various cancer types. Methods: Colorectal cell line LIM1215, SW48 and NSCLC cell line NCI-H1975, NCI-H838 were used for MTT assays. Apoptosis assay was performed using the annexin V-FITC apoptosis detection kit (BioBud, Cat. LS-02-100). Colony forming assay was performed with 6-well culture plates by seeding 500 cells/well. The expression levels of proteins involved in Q2a-induced signaling pathway were determined by Western blot. In vivo study was performed with tumor xenografts. HT-29 cells were implanted into the right flank of 5-week-old BALB/c nude mice. Tumor size was determined by measuring the diameter (width, length) of the tumor every two days with a digital caliper. All group mice sacrificed at day 19. Results: Patients with cancer involving KRAS mutations are resistant to therapeutics that target epidermal growth factor receptor and show poor outcomes. In addition, effective treatments that target G12D in KRAS have not been developed. Thus, therapeutic agents targeting the G12D mutation in KRAS are needed. In this study, we identified the Q2a compound, a quinazoline-based derivative that specifically acts on the KRAS G12D mutation. This compound induced cell apoptosis by reducing phosphorylation of AKT in the PI3K pathway downstream of KRAS. Q2a compound specifically inhibited the growth of KRAS G12D mutant cells. In addition, cells with the KRAS G12D mutation were more sensitive to Q2a compound compared to the sensitivity of wild-type cells and those with other KRAS mutations, resulting in apoptosis. Treatment with the Q2a compound also decreased C-RAF phosphorylation in the MAPK pathway, which is downstream of KRAS. In KRAS G12D-mutated colorectal cancer xenografts, Q2a compound inhibited tumor production without significant liver toxicity. Conclusion: Q2a compounds show potential as therapeutic agents that target KRAS G12D mutations in colorectal and pancreatic cancers. Kyewords: Anti-Cancer Drug, KRAS G12D Mutant, apoptosis, colon cancerJi Yoon Lee, Dae-Hee Lee*Department of Marine Bio Food Science, Gangneung-Wonju national University Citation Format: Dae-Hee Lee, Ji Yoon Lee. Inhibition of KRAS G12D mutant with small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5770.

Abstract 554: Determining the role of PROX1 in prostate cancer neuroendocrine trans-differentiation

Abstract Nonbiased mutational screening to identify essential genes and pathways in cancer is a powerful approach for understanding underlying biology as well as the identification of potential therapeutic targets. Epigenetic (EPI) and transcriptional factors (TF) play an important role in the change of phenotype and lineage plasticity from adenocarcinoma to castration resistant prostate cancer (CRPC), double negative (DN) or treatment-induced neuroendocrine prostate cancer (NEPC). When RB1 loss occurs in combination with TP53 alterations, stemness and lineage plasticity increase with one consequence being differentiation to a neuroendocrine (NE) lineage. Here, we are identifying and testing the functional role of epigenetic and transcription factors required for the viability of CRPC, DN and NEPC relative to a matrix of genotypic and phenotypic characteristics. We applied CRISPR-CAS9 dropout screens in prostate organoids and cell lines to identify gene/s associated with CRPC, DN and NEPC viability. We successfully CRISPR-engineered LNCaP clones of single (LNCaP-TP53-SKO, LNCaP-RB1-SKO) and double (LNCaP-TP53-RB1-DKO) gene knockouts, which were subsequently characterized for immuno-phenotype and transcriptome. Our dropout screens, targeting all TFs and EPIs, identified 117 TF and 40 EPI genes responsible for the viability of DN, NEPC and LNCaP models. We found known genes such as FOXA1 and ASCL1, as well as novel genes including PROX1 and KLF3, which were validated through guide RNA competition assays. Our results showed PROX1 is essential for cell viability in a DN organoid model, and its expression is associated with increased aggressiveness, indicating PROX1 has role in stemness/lineage plasticity in treatment resistant adenocarcinoma. RNA-seq analysis following overexpression of PROX1 in the LNCaP-TP53-RB1 null adenocarcinoma model, with and without enzalutamide, identified pathways enriched for cell proliferation, differentiation, neuronal development, and prostate gland development. In summary, our study has suggested an important role for PROX1 in the growth and differentiation of poorly differentiated prostate cancer, which may lead to the identification of potential therapeutic targets. Citation Format: Sonam Raj, Brian Capaldo, Joel Bowman, Margaret White, Xavier Moore, Berkley Gryder, Aian N. Alilin, David Takeda, Kathleen Kelly. Determining the role of PROX1 in prostate cancer neuroendocrine trans-differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 554.

Endothelial LAT1 (SLC7A5) Mediates S-Nitrosothiol Import and Modulates Respiratory Sequelae of Red Blood Cell Transfusion In Vivo.

Increased adhesivity of red blood cells (RBCs) to endothelial cells (ECs) may contribute to organ dysfunction in malaria, sickle cell disease, and diabetes. RBCs normally export nitric oxide (NO)-derived vascular signals, facilitating blood flow. S-nitrosothiols (SNOs) are thiol adducts formed in RBCs from precursor NO upon the oxygenation-linked allosteric transition in hemoglobin. RBCs export these vasoregulatory SNOs on demand, thereby regulating regional blood flow and preventing RBC-EC adhesion, and the large (system L) neutral amino acid transporter 1 (LAT1; SLC7A5) appears to mediate SNO export by RBCs. To determine the role of LAT1-mediated SNO import by ECs generally and of LAT1-mediated SNO import by ECs in RBC SNO-dependent modulation of RBC sequestration and blood oxygenation in vivo, we engineered LAT1fl/fl; Cdh5-Cre+ mice, in which the putative SNO transporter LAT1 can be inducibly depleted (knocked down, KD) specifically in ECs ("LAT1ECKD"). We show that LAT1 in mouse lung ECs mediates cellular SNO uptake. ECs from LAT1ECKD mice (tamoxifen-induced LAT1fl/fl; Cdh5-Cre+) import SNOs poorly ex vivo compared with ECs from wild-type (tamoxifen-treated LAT1fl/fl; Cdh5-Cre-) mice. In vivo, endothelial depletion of LAT1 increased RBC sequestration in the lung and decreased blood oxygenation after RBC transfusion. This is the first study showing a role for SNO transport by LAT1 in ECs in a genetic mouse model. We provide the first direct evidence for the coordination of RBC SNO export with EC SNO import via LAT1. SNO flux via LAT1 modulates RBC-EC sequestration in lungs after transfusion, and its disruption impairs blood oxygenation by the lung.

Abstract 7418: LongFuse: Detecting gene fusion transcripts from high throughput long-read single cell RNA sequencing data

Abstract Gene fusion resulted from genomic translocation plays important oncogenic roles including but not limited to gene activation and changing protein coding sequences. Historically, fusion gene analysis has been limited to bulk levels resulting in mixed oncogene activation models. Single cell Nanopore RNA sequencing (scNanoRNAseq) has the unique strength in detecting full-length chimeric transcripts in thousands of single cells in parallel. However, robust computational tools for detecting fusion genes in single cells from scNanoRNAseq data are missing. Here, we developed LongFuse, a fast and accurate tool, to detect fusion genes and their splicing isoforms in single cells from scNanoRNAseq data. LongFuse implements an XOR logic gate algorithm to speed up fusion candidate searching and stringent criteria to eliminate false discoveries due to trans-splicing events and randomly ligated chimeras. We applied LongFUSE onto scNanoRNAseq data of both human prostate cancer cell line, VCaP, and tumor tissues with known TMPRSS2-ERG fusion. Our results showed that both cancer cell line and prostate tumors harbored a mixture of fusion positive and fusion negative tumor cells. Gene expression analysis revealed transcriptional differences between fusion positive and negative cells including genes involved in cancer cell proliferation and invasion programs such as ELL2, ZBTB16, IGF1R, ANK3, MYPOP and PLPP1 genes. LongFUSE also calculates the splicing isoforms of fusion gene transcripts, allowing prediction of protein sequences of fusion gene transcripts. Taken together, our data demonstrated that LongFUSE is a robust computational tool for detecting gene fusions and their splicing isoforms at single cell level from scNanoRNAseq data. Citation Format: Cheng-Kai Shiau, Yueying He, Hsiao-Yun Lin, Lina Lu, Xiao-Dong Lu, Jindan Yu, Ruli Gao. LongFuse: Detecting gene fusion transcripts from high throughput long-read single cell RNA sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7418.

Abstract 3787: Important role of GPX1 and NF-kb signaling pathway in human gastric cancer including cell proliferation and invasion

Abstract Background: Glutathione peroxidases(GPXs) are member of an antioxidant enzyme family. Their role is to eliminate the reactive oxygen species(ROS). Many available studies confirm that over expression of GPXs can promote cancer cell proliferation and invasion. Among the 8 GPXs, Glutathione peroxidase-1 (GPX1) is the most plentiful. Overexpression of GPX1 has been noted to promote invasion, migration, cisplatin resistance and proliferation in breast, colorectal and lung cancer. NF-kB is a ubiquitous nuclear transcription factor in the human body. that key role in cell proliferation, apoptosis and tumor progression. In reliable studies, inhibition of NF-kB expression has been shown to reduce the malignancy of Esophageal squamous cell carcinoma. Accordingly, the purpose of this study is to identify the GPX1 and NF-kB signaling pathway and their correlation with cancer cell proliferation and invasion in vitro using gastric cancer cell. Methods: In our study, we used cell culture, Northern blotting, cDNA microarray analysis, western blotting, RT-PCR, Zymography, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, GPX1 knock-down with short hairpin RNS(shRNA), Standard two-chamber invasion assay, Chromatin immunoprecipitation assay. Results: First, we confirmed that the expression level of GPX1 was up-regulated by HGF(hepatocyte growth factor) in gastric cancer cells. The cells were treated with NF-kB inhibitor, Pyrrolidine dithiocarbamate(PDTC), and analyzed by Western blotting to identify the associated pathway of HGF-induced GPX1 with NF-kB. The HGF-induced GPX1 protein level was down regulated by PDTC. The role of GPX1 associated with NF-kB was determined by knock down GPX1 expression using GPX1-sh RNA. The GPX1-sh RNA treated cells showedvdown regulation of NF-kB and urokinase Plasminogen activator(uPA) also. To fine the upper regulator of GPX1 signaling pathway, we treated AKT inhibitor(LY294002) ant then analyzed by Western blotting to identify associated pathway of HGF-induced GPX1 with AKT. When AKT pathway was inhibited, GPX1 and NF-kB were down-regulated in gastric cancer cell. HGF-mediated cell proliferation and invasion in vitro was decreased in GPX1 knock down cell. We identified the putative binding site of GPX1 promotor containing NF-kB binding site and confirmed the function by CHIP assay. Conclusions: This study aimed to investigate relationship between GPX1 and NF-kB in gastric cancer cells. Results showed that HGF induced GPX1 expression through the NF-kB and AKT pathway and GPX1 may play a significant role in cell proliferation and invasion in gastric cancer. Therefore, GPX1 can be a potential therapeutic target of gastric cancer for the treatment. Citation Format: Byeong Il Jang, Ji Yoon Jung, Sung Ae Koh, Kyung Hee Lee. Important role of GPX1 and NF-kb signaling pathway in human gastric cancer including cell proliferation and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3787.

Abstract 307: Generation and characterization of a glioblastoma stem cell model with acquired resistance to LSD1 inhibition

Abstract Epigenetic modifications play an important role in the initiation of various cancer types, including glioblastoma (GBM), and in the development of therapeutic resistance. Patients with GBM, a common malignant brain tumor, have an average survival of 12-15 months after first-line therapy. Lysine-specific demethylase 1 (LSD1) is a histone demethylase that is overexpressed in glioblastoma stem cells (GSCs) and functions to promote proliferation, inhibit differentiation, and enhance motility. Previous work from our group has investigated LSD1 as a therapeutic target in glioblastoma and identified genes that may predict resistance to pharmacological LSD1 inhibitors using RNA-seq. Five upregulated genes, HKDC1, RAB3IL1, RAB39B, FTH1, and FAM213A, were associated with LSD1 inhibitor (LSD1i) resistance and were validated in brain tissue of a GBM model treated with GSK-LSD1 after succumbing to tumor burden. In order to determine whether selection pressure from LSD1 inhibitor treatment caused upregulation of these genes, we created a model of acquired resistance in GSC lines in vitro. Isogenic resistant lines were generated by treating MDA-GSC17 cells with increasing concentrations of LSD1i or diluent (DMSO), as tolerated starting at 50 uM (which was five fold below the IC50 for these lines). In parallel, MDA-GSC17 controls were exposed to DMSO. The resistant line was created over 4 weeks and monitored by cell number and viability to reach 90%. To assess the change in gene expression, we conducted quantitative PCR for the five genes in the parental and resistant MDA-GSC17 line and saw upregulation in 3 of the 5 genes. Two genes, HKDC1 and RAB3IL1, were unable to be assessed due to low basal expression. However, 3 genes were upregulated which are RAB39B, FTH1, and FAM213A. RAB39B is a Ras-analog in brain (RAB) small GTPase and a member of the RAS oncogene family, involved in vesicular trafficking. High expression of ferritin heavy chain 1 (FTH1) is correlated with high-grade gliomas and poor prognosis in glioblastoma. Lastly, the redox regulatory protein, FAM213A, can activate antioxidant proteins and elevated expression is associated with worse outcomes for AML patients. This suggests that modulating the RAS signaling pathways and specific redox pathways may overcome LSD1 resistance. Understanding the changes in gene expression as the cells acquire resistance to LSD1 inhibitors will help us understand the mechanism of resistance to LSD1 inhibitors in glioblastoma and to design new treatment strategies. Citation Format: Kareena H. Patel, Lea Stitzlein, Deokhwa Nam, Frederick Lang, Joy Gumin, Joya Chandra. Generation and characterization of a glioblastoma stem cell model with acquired resistance to LSD1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 307.

Abstract 4409: FBXW7 and BCL9L altered pathways as hallmarks of early-onset colorectal cancer

Abstract Background: The incidence of early-onset colorectal cancer (EO-CRC) has been alarmingly increasing, particularly among non-Hispanic Whites (NHWs), resulting in similar rates to ones in African Americans (AAs). Overall, the median age of diagnosis has decreased from 72 to 66 years. The molecular features that could potentially distinguish this phenotype are still not well-established. This study aimed to identify genetic, epigenetic, and transcriptomic factors associated with EO-CRC. Methods: 510 patients with microsatellite-stable tumors analyzed for somatic mutations, targeting 20 cancer driver genes. We included a cohort of 175 AAs from the Yale New Haven Health System, a previously studied cohort of 51 AAs from the Chicago Colorectal Cancer Consortium, and the publicly available molecular data from TCGA COAD-READ PanCancer cohort (50 AAs and 229 NHWs). We analyzed the transcriptomic and methylation profiles from the Yale cohort using Tempo-SeqTM and Illumina EPIC array, respectively. Results: Of the 510 patients, 18% were diagnosed with EO-CRC. Using a multiple variant analysis, adjusting for sex, race, tumor location, and stage, EO-CRC patients were less likely to be male (OR = 0.59, 95% CI: 0.37-0.94, p-value = 0.03), their tumors lacked mutations in APC (OR = 0.37, 95% CI: 0.22-0.63, p-value = 0.0002), and they were more likely to have mutations in FBXW7 (OR = 3.1, 95% CI: 1.60-6.00, p-value = 0.0007) and BCL9L (OR = 3.1, 95% CI: 1.00-8.72, p-value = 0.04). We compared tumors with and without mutations in FBXW7 or BCL9L in two independently analyses. Combining transcriptomics and methylation data, we identified 10 candidate genes that showed significant gene downregulation with hypermethylation or upregulation with hypomethylation. Among FBXW7 mutant tumors, we found the Cartilage oligomeric matrix protein (COMP) gene was significantly downregulated and hypermethylated. In CRC, COMP has been associated with a crosstalk between the TGF-β pathway and immune cell infiltration. Moreover, inactivation of FBXW7 resulted in the accumulation of phosphorylated TGIF1 molecules and repression of TGFβ-dependent transcription in cancer cell lines. Deficiency of FBXW7 is a key element in the phosphorylation-dependent ubiquitination and subsequent proteasome degradation of oncoproteins, such as c-MYC, NOTCH, and Cyclin E. Among BCL9L mutant tumors, keratin 20 (KRT20), the intermediate filament protein and marker of intestinal differentiation in CRC, was downregulated and hypermethylated. BCL9L plays an important role in tumorigenesis induced by aberrant Wnt signaling or aneuploidy tolerance. Conclusion: Our findings further demonstrate the importance of specific etiologic mechanisms in EO-CRC at the level of somatic alterations, providing new ways to understand the biology of EO-CRC and prospects for the development of targeted therapies. Citation Format: Reger Mikaeel, Dante Bellomo, Seeta Rajpara, David Buckley, Mary Yagle, Baris Kerimoglu, Curtis Thorne, Megha Padi, Bodour Salhia, Nathan A. Ellis, Xavier Llor, Rosa Xicola. FBXW7 and BCL9L altered pathways as hallmarks of early-onset colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4409.

Roles of IL-4, IL-13, and Their Receptors in Lung Cancer.

Interleukin (IL)-4 and IL-13 are the main effectors of innate lymphoid cells (ILC2) of the type 2 innate immune response, which can carry out specific signal transmission between multiple cells in the tumor immune microenvironment. IL-4 and IL-13 mediate signal transduction and regulate cellular functions in a variety of solid tumors through their shared receptor chain, the transmembrane heterodimer interleukin-4 receptor alpha/interleukin-13 receptor alpha-1 (type II IL-4 receptor). IL-4, IL-13, and their receptors can induce the formation of a variety of malignant tumors and play an important role in their progression, growth, and tumor immunity. In order to explore possible targets for lung cancer prediction and treatment, this review summarizes the characteristics and signal transduction pathways of IL-4 and IL-13, and their respective receptors, and discusses in depth their possible role in the occurrence and development of lung cancer.

Abstract 4434: Cancer cell-derived adrenomedullin downregulates Zeb1 in adipocytes to induce adipolysis and promote breast cancer progression

Abstract Background: Once breast carcinoma in situ breaks through the basement membrane, it preferentially invades the lipid-rich microenvironment around the mammary gland, which indicates a poor prognosis. However, the detailed mechanisms of this interaction remain elusive. Here, we describe bidirectional communication between breast cancer cells and cancer-associated adipocytes (CAAs). Different from the common hormone-driven mechanism of lipolysis of adipocytes, we found that CAAs perform a unique lipolysis program, and Zeb1 plays an important role in this lipolysis process. Methods: Fabp4-cre;Zeb1-/- as adipose tissue-specific Zeb1 gene knockout homozygous mice and Fabp4-cre;Zeb1 tg as adipose tissue-specific Zeb1 gene overexpression mice were constructed to study mammary gland development and perform orthotopic tumor transplantation experiments. Tetracycline-induced Zeb1-overexpressing stable cells were constructed from mouse preadipocytes 3T3-L1 to study phenotypes of lipolysis. Single-cell sequencing, targeted lipidomics, and RNA sequencing analysis were used to investigate the interaction between cancer cells and adipocytes. Western blotting and immunohistochemical staining assays were used to detect the expression Zeb1 at the cellular level, in mouse models and in patient tissue samples. Results: Our results indicated that breast cancer cell-derived adrenomedullin (ADM) significantly down-regulates Zeb1 expression in adipocytes, which drives downstream gene expression of Pnpla2/HSL/SCD1 and induces adipocyte lipolysis into CAA, a process that results in the release of excess palmitoleic acid. Consequently, we proved that palmitoleic acid could replace arachidonic acid (ARA) in the phospholipid sn-2 position of breast cell membrane, which enhances the fluidity and invasiveness of cancer cells. In addition, overexpression of Zeb1 in adipocytes reduces the ability of cancer cells to induce adipolysis and inhibits the malignant progression of cancer cells. Conclusion: These results indicate that Zeb1 plays a pivotal role as core transcriptional factor in CAA lipolysis. The interaction between breast cancer cells and adipocytes leads to the reprogramming of metabolism within their tumor microenvironment, which may become a new target for breast cancer treatment. Citation Format: Lixia Cao, Xiao Chen, Yi Shi, Shuang Yang. Cancer cell-derived adrenomedullin downregulates Zeb1 in adipocytes to induce adipolysis and promote breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4434.

Can intellectual property rights pilots reduce carbon emissions? Evidence from China

In this paper, panel data from Chinese cities from 2000 to 2020 were used, and the intellectual property pilot was used as a quasi-natural experiment. The double difference model mainly tests the underlying mechanism of the impact of intellectual property rights pilots on urban carbon emissions. This study revealed that IPR pilots play an important role in reducing urban carbon emissions. Heterogeneity experiments reveal that IPR pilots are conducive to reducing carbon emissions in coastal as well as high greening cities and that the underlying mechanism through which IPR pilots affect urban carbon emissions mainly occurs at the level of innovation and the optimization of the industrial structure. In addition, this paper also validates the smoothness of the model by replacing the explanatory variables and performing a placebo test, which further strengthens the reliability of the paper’s conclusions.

Abstract 412: Discovery and preclinical characterization of ISM9342A, a novel, potent, and orally bioavailable WRN inhibitor that suppresses MSI-H tumor growth

Abstract Loss of function mutations in DNA mismatch repair genes can result in microsatellite instabilities (MSI) and high levels of MSI (MSI-H) contributes to tumorigenesis and is frequently observed in endometrial carcinoma, gastric cancer, and colorectal cancer. Although immune checkpoint inhibitors have been approved for the treatment of MSI-H tumors, there is still a large proportion of patients with MSI-H tumors who fail to respond to these therapies. Recently, two independent genome-wide loss of function screens have identified Werner syndrome helicase (WRN) as the top dependency for MSI-H cancer cell viability. WRN plays important roles in DNA repair and in the maintenance of genome integrity. Given that WRN deficiency selectively impairs the viability of MSI-H but not microsatellite stable cancer cells, WRN is a synthetic lethal target for MSI-H tumors. ISM9342A is a novel and potent WRN inhibitor exhibiting single digit nanomolar IC50 values in both ATPase and helicase assays. ISM9342A suppressed the proliferation and increased double strand break markers in MSI-H cancer cells. Moreover, ISM9342A demonstrated promising in vivo anti-tumor efficacy in an MSI-H xenograft model along with tumor regression. ISM9342A demonstrated good drug-like properties, including excellent in vitro ADMET profiles and good in vivo exposure, including low clearance and optimal oral bioavailability across multiple preclinical species. Collectively, these findings highlight the potential of ISM9342A as a highly potent WRN inhibitor for the treatment of MSI-H tumors Citation Format: Pei Zhao, Wei Zhu, Xiaoyu Ding, Jianfei Wan, Xiaojing Chen, Man Zhang, Xiao Ding, Supriya Bavadekar, Sujata Rao, Feng Ren, Alex Zhavoronkov. Discovery and preclinical characterization of ISM9342A, a novel, potent, and orally bioavailable WRN inhibitor that suppresses MSI-H tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 412.

Abstract 5682: Micro RNA level changes in prostate cancer cells treated with MDMX and MDM2 inhibitors

Abstract INTRODUCTION: Prostate Cancer (PCa) is the most common non-cutaneous cancer and the second leading cause of cancer death among men in the United States. Most of the PCa cases are diagnosed in men between the ages of 65-74. Due to the recent push for frequent prostate screenings, these cancers are being detected early and treated much faster, leading to a 5-year survival of a localized or regional PCa to >99%. However, the 5-year survival of PCa with distant metastasis is significantly reduced to 32%, especially with involvement of osteoblastic bone lesions, lung lesions, and brain lesions. Therefore, there is a clinical need to identify prognostic markers and new targets for developing therapies and effectively treating advanced PCa. Previous studies have shown that levels of MDMX and MDM2, oncogenic inhibitors of p53 tumor suppressor protein, are significantly elevated in PCa cells. Inhibition of MDM2 and MDMX using specific inhibitors has been shown to elevate the levels of p21, p27, and p53 in LNCaP (Lymph Node Carcinoma of the Prostate) cells leading to cell death. Recent studies have demonstrated that micro RNAs (miRNAs) play an important role in supporting the tumor suppressor function of p53 through regulating the balance between p53 and MDM2. In this regard, several miRNAs were found to be significantly altered to modulate the level and intracellular mechanisms of p53 and MDM2 in various cancers. OBJECTIVES: To determine the relative levels of miRNA31, miRNA-34a and miRNA-128 in LNCaP cells that were treated with NSC-207895 (MDMX inhibitor), SJ-172550 (MDMX inhibitor), and RG-7388 (MDM2 inhibitor). METHODS: LNCaP cells were treated with NSC-207895 (20 uM), SJ-172550 (20 uM) or RG-7388 (2 uM) for 24 hrs. After completing the treatments, the miRNAs were extracted from the LNCaP cells using the miRNeasy Tissue/Cells Advanced Kit from Qiagen. We analyzed the levels of miRNA-31, miRNA-34a, and miRNA-128 in the treated samples using qRT-PCR analysis method and compared the levels to the control. During the qRT-PCR analysis the cDNA was amplified using Meridian Biosciences qPCR Master Mix and specific primers for miRNAs from Origene were used according to the manufacturer's protocol. RESULTS: Our experimental results indicate that inhibition of MDMX using NSC-207895 and SJ-172550 can elevate the levels of p21, p27, and p53 leading to cell death. Determination of cell death markers in the past revealed induction of necroptosis following drug treatments. Our current results indicate measurable changes in the levels of miRNA-31 and miRNA-34a following inhibition of MDMX using SJ-172550. We are in the process of determining the possible link between the changes in the levels of miRNAs and cell death. ACKNOWLEDGEMENT: This research was supported by the Royal Dames of Cancer Research, Ft. Lauderdale, Florida. Citation Format: Rojin Fatirkhorani, Ahmed Saleh Alsarrani, Shyam Sundar Jaganathan, Umamaheswari Natarajan, Appu Rathinavelu. Micro RNA level changes in prostate cancer cells treated with MDMX and MDM2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5682.

Abstract 7563: Efficacy of JPI-547, novel dual PARP inhibitor, in endometrial cancer cell line

Abstract Objectives: PARP inhibitor is effective in treating DNA repair deficiency endometrial cancer. Endometrial cancer (EC) is one of the most common gynecological cancers in developed countries and its incidence is increasing, and the prognosis of patients with early stage and low-risk EC is very good, but the prognosis of progressive and recurrent EC is poor. Novel PARP inhibitor JPI-547 is a PARP and tankyrase dual inhibitor, and in cancer cells, tankyrase inhibitor plays an important role in DNA repair, telomere maintenance, and cell survival. This experiment was conducted to determine whether the reactivity of the PTEN mutant EEC was increased by using the Novel PARP inhibitor. Method: Human endometrioid endometrial cancer cell lines, Hec-1A, Hec-1B and Ishikawa were obtained. Cell viability assay for the drugs Olaparib and JPI-547 were assessed. Colonies consisting of at least 50 cells were counted. The number of dead cells was counted in five different areas of the hemocytometer, and the experiment was performed three times. The percentage of dead cells was analyzed compared to the untreated group. We confirmed the efficacy by siRNA transfection and immunofluorescence. Result: When treated with Olaparib, there was no change in cell viability in the rest of the cells except for Ishikawa cells, which are PTEN mutations. It decreased by 35.4% in Ishikawa cells, whereas when treated with JPI-547, all cell lines Hec-1A, Hec-1B, and Ishikawa cell viability decreased by 28%, 34.3%, and 48.7%, respectively. PTEN knock-down reduced cell visibility by 28% when treated with olaparib, whereas when treated with JPI-547, Hec-1A (siCont) and Hec-1A (siPTEN) decreased by 26% and 42%, respectively, in all cells regardless of whether PTEN knock-down is present. When treated with Olaparib, dead cells were identified only in PTEN knock-down cells, increasing by 22%. When treated with JPI-547, dead cells increased by 25.5% and 33% in all cells of Hec-1A (siCont) and Hec-1A (siPTEN), respectively. It was confirmed that JPI-547 can reduce cell viability regardless of the formation of Rad51 according to PTEN. Conclusion: JPI-547, a novel PARP inhibitor, showed good efficacy in PTEN mutation and PTEN-wild-type EEC cell lines than in conventional PARP inhibitor. The effect of reducing survival of ECC cell line with JPI-547 was confirmed with or without PTEN mutation. Further evaluation and in vivo experiments are needed to prove the efficacy of JPI-547 in EC. Citation Format: Shin-Wha Lee, So-Hyun Nam, Su-Bin Park. Efficacy of JPI-547, novel dual PARP inhibitor, in endometrial cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7563.

Abstract 6250: The RNA editing landscape of lung cancer in never smokers

Abstract Adenosine-to-Inosine (A-to-I) editing represents a crucial RNA modification mechanism, holding the potential to drive carcinogenesis. This sophisticated post-transcriptional process is catalyzed by a series of ADAR (adenosine deaminase acting on RNA) family members, which can profoundly reshape the cellular epitranscriptomic landscape. While previous explorations have illuminated the important role of A-to-I editing in lung cancer tumorigenesis and prognosis, most studies have focused on lung cancer from smokers. To investigate the contribution on RNA-editing in lung cancers in never smokers (LCINS) , we embarked on a rigorous dissection of A-to-I RNA editing events in 603 LCINS. Harnessing transcriptome and whole-genome sequencing technologies, we identified 15,880 A-to-I sites, of which 75% were positively linked to the enzymatic activity of ADAR1. 92.80% of detected editing sites were located at Alu repetitive regions, with the remainder in non_Alu repeat (4.1%) and non-repeat regions (3.1%). The global RNA editing levels, measured as Alu Editing Index (AEI), were found to be significantly associated with ADAR1 expression, Tumor Mutational Burden, and Neoantigens Burden. A subtle correlation between global RNA editing levels and tumor stage was also noted. Delving into cancer-associated editing events, we identified 5,539 differentially edited sites in tumors compared with normal tissues, which were located at 823 genes, including several key tumor suppressors and oncogenes, like MDM2, BRAF, TP53, BRCA2, ATM, and VHL. Subsequent pathway analysis identified TGF-beta, Notch Signaling, and DNA Damage Response as the key pathways for cancer progression. Using the characterized A-to-I editing profiles, we built a prognostic risk stratification model, with high risk scores significantly linked to poor survival outcomes, even after adjusting for other clinical factors. Our study provides a comprehensive A-to-I editing landscape of lung cancer in never smokers, highlighting the potential roles of RNA modifications in the pathogenesis of this lethal disease. Citation Format: Jian Sang, Wei Zhao, Tongwu Zhang, Maria Teresa Landi. The RNA editing landscape of lung cancer in never smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6250.