Compared Gene Expression Research Articles

A Novel Gene and Comparison of Gene Expressions in Low- and High-Grade Renal Cell Carcinomas

A Novel Gene and Comparison of Gene Expressions in Low- and High-Grade Renal Cell Carcinomas

Responding to ACL Injury and its Treatments: Comparative Gene Expression between Articular Cartilage and Synovium.

The relationship between cartilage and synovium is a rapidly growing area of osteoarthritis research. However, to the best of our knowledge, the relationships in gene expression between these two tissues have not been explored in mid-stage disease development. The current study compared the transcriptomes of these two tissues in a large animal model one year following posttraumatic osteoarthritis induction and multiple surgical treatment modalities. Thirty-six Yucatan minipigs underwent transection of the anterior cruciate ligament. Subjects were randomized to no further intervention, ligament reconstruction, or ligament repair augmented with an extracellular matrix (ECM) scaffold, followed by RNA sequencing of the articular cartilage and synovium at 52 weeks after harvest. Twelve intact contralateral knees served as controls. Across all treatment modalities, the primary difference in the transcriptomes was that the articular cartilage had greater upregulation of genes related to immune activation compared to the synovium-once baseline differences between cartilage and synovium were adjusted for. Oppositely, synovium featured greater upregulation of genes related to Wnt signaling compared to articular cartilage. After adjusting for expression differences between cartilage and synovium seen following ligament reconstruction, ligament repair with an ECM scaffold upregulated pathways related to ion homeostasis, tissue remodeling, and collagen catabolism in cartilage relative to synovium. These findings implicate inflammatory pathways within cartilage in the mid-stage development of posttraumatic osteoarthritis, independent of surgical treatment. Moreover, use of an ECM scaffold may exert a chondroprotective effect over gold-standard reconstruction through preferentially activating ion homeostatic and tissue remodeling pathways within cartilage.

RNA profiles to distinguish multiple sclerosis (MS) and neuromyelitis optica (NMO) in peripheral whole blood (S9.003)

RNA profiles to distinguish multiple sclerosis (MS) and neuromyelitis optica (NMO) in peripheral whole blood (S9.003)

Biological roles of myeloid cell subsets during CNS inflammation (P2-3.005)

<h3>Objective:</h3> We designed an experimental platform, utilizing single-cell transcriptomic and epigenomic profiling. We employed this system to interrogate single-cell data for subset-specific signatures. <h3>Background:</h3> Myeloid cells (MC) of the central nervous system (CNS) constitute a heterogeneous population that includes A) yolk sac-derived CNS microglia, B) bone marrow-derived resident MC and C) infiltrating bone marrow-derived MC. While bone marrow and yolk sac progenies differ in origin, their protein expression exhibit significant overlap. Current canonical myeloid markers, lack the specificity required for use as disease biomarkers or therapeutic targets. <h3>Design/Methods:</h3> We created chimeras in congenic animals by transplantation of bone marrow from CD45.1⁺ donors into sub-lethally irradiated CD45.2⁺ recipients. Following active EAE, CD11b⁺ MC were collected from CNS at the peak of the disease. Fluorescent-activated cell sorting (FACS) separated CD45.1⁺ MC from CD45.2⁺ MC. Each sample was analyzed simultaneously for single-cell gene expression (scRNA-seq) and epigenetic signatures (scATAC-seq) via the 10X Genomics Multiomic assay. We employed bioinformatics software (Seurat v4.0) for analysis and visualization of data profiling over 20,000 cells. <h3>Results:</h3> Bone marrow derived (CD45.1+) or microglial and CNS-resident (CD45.2+) MC were both present in the CNS tissue during acute EAE. Analyses of single-cell transcriptomic and ATAC-seq data, independently distinguished common clusters of MC derived entirely from the CD45.2+ cells. Unsupervised annotation, recognized these cells as microglia. CD45.1+ MC and microglia exhibited extensive gene expression overlap; however, differential gene expression analysis and logistic regression modeling yielded a signature set of genes within the microglia accurately distinguished microglia from bone-marrow-derived MC in independent datasets. <h3>Conclusions:</h3> Single-cell data recapitulates the subtle differences in the otherwise comparable gene expression patterns among microglia and bone marrow-derived MC. These patterns shed light on the subset-specific biological roles and may present as superior substitutes for the suboptimal canonical myeloid markers. <b>Disclosure:</b> Navid Manouchehri has nothing to disclose. The institution of an immediate family member of Dr. Salinas has received research support from Veterans Health Administration. Rehana Hussain has nothing to disclose. Dr. Stuve has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for EMD Serono. Dr. Stuve has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for Novartis. Dr. Stuve has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for Roche Genentech. Dr. Stuve has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for TG Therapeutics. Dr. Stuve has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for TG Therapeutics. Dr. Stuve has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for EMD Serono. Dr. Stuve has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for VYNE. Dr. Stuve has received personal compensation in the range of $5,000-$9,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Therapeutic Advances in Neurological Diseases. Dr. Stuve has received research support from US Department of Veterans Affairs. Dr. Stuve has received research support from National Multiple Sclerosis Society (US). Dr. Stuve has received research support from Exalys. Dr. Stuve has received research support from Merck KGaA.

Transcriptomic Comparison of Human Peripartum and Dilated Cardiomyopathy Identifies Differences in Key Disease Pathways

Peripartum cardiomyopathy (PPCM) is a rare form of acute onset heart failure that presents in otherwise healthy pregnant women around the time of delivery. While most of these women respond to early intervention, about 20% progress to end-stage heart failure that symptomatically resembles dilated cardiomyopathy (DCM). In this study, we examined two independent RNAseq datasets from the left ventricle of end-stage PPCM patients and compared gene expression profiles to female DCM and non-failing donors. Differential gene expression, enrichment analysis and cellular deconvolution were performed to identify key processes in disease pathology. PPCM and DCM display similar enrichment in metabolic pathways and extracellular matrix remodeling suggesting these are similar processes across end-stage systolic heart failure. Genes involved in golgi vesicles biogenesis and budding were enriched in PPCM left ventricles compared to healthy donors but were not found in DCM. Furthermore, changes in immune cell populations are evident in PPCM but to a lesser extent compared to DCM, where the latter is associated with pronounced pro-inflammatory and cytotoxic T cell activity. This study reveals several pathways that are common to end-stage heart failure but also identifies potential targets of disease that may be unique to PPCM and DCM.

Computational screen to identify potential targets for immunotherapeutic identification and removal of senescence cells.

To prioritize gene and protein candidates that may enable the selective identification and removal of senescent cells, we compared gene expression signatures from replicative senescent cells to transcriptomics and proteomics atlases of normal human tissues and cell types. RNA-seq samples from in vitro senescent cells (6 studies, 13 conditions) were analyzed for identifying targets at the gene and transcript levels that are highly expressed in senescent cells compared to their expression in normal human tissues and cell types. A gene set made of 301 genes called SenoRanger was established based on consensus analysis across studies and backgrounds. Of the identified senescence-associated targets, 29% of the genes in SenoRanger are also highly differentially expressed in aged tissues from GTEx. The SenoRanger gene set includes previously known as well as novel senescence-associated genes. Pathway analysis that connected the SenoRanger genes to their functional annotations confirms their potential role in several aging and senescence-related processes. Overall, SenoRanger provides solid hypotheses about potentially useful targets for identifying and removing senescence cells.

Classification related to immunogenic cell death predicts prognosis, immune microenvironment characteristics, and response to immunotherapy in lower-grade gliomas.

Immunogenic cell death (ICD) is a form of cell death that elicits immune responses against the antigens found in dead or dying tumor cells. Growing evidence implies that ICD plays a significant role in triggering antitumor immunity. The prognosis for glioma remains poor despite many biomarkers being reported, and identifying ICD-related biomarkers is imminent for better-personalized management in patients with lower-grade glioma (LGG). We identified ICD-related differentially expressed genes (DEGs) by comparing gene expression profiles obtained across Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) cohorts. On the foundation of ICD-related DEGs, two ICD-related clusters were identified through consensus clustering. Then, survival analysis, functional enrichment analysis, somatic mutation analysis, and immune characteristics analysis were performed in the two ICD-related subtypes. Additionally, we developed and validated a risk assessment signature for LGG patients. Finally, we selected one gene (EIF2AK3) from the above risk model for experimental validation. 32 ICD-related DEGs were screened, dividing the LGG samples from the TCGA database into two distinct subtypes. The ICD-high subgroup showed worse overall survival (OS), greater immune infiltration, more active immune response process, and higher expression levels of HLA genes than the ICD-low subgroup. Additionally, nine ICD-related DEGs were identified to build the prognostic signature, which was highly correlated with the tumor-immune microenvironment and could unambiguously be taken as an independent prognostic factor and further verified in an external dataset. The experimental results indicated that EIF2AK3 expression was higher in tumors than paracancerous tissues, and high-expression EIF2AK3 was enriched in WHO III and IV gliomas by qPCR and IHC, and Knockdown of EIF2AK3 suppressed cell viability and mobility in glioma cells. We established novel ICD-related subtypes and risk signature for LGG, which may be beneficial to improving clinical outcome prediction and guiding individualized immunotherapy.

Transcriptome analysis reveals steroid hormones biosynthesis pathway involved in abdominal fat deposition in broilers

Excessive abdominal fat deposition reduces the feed efficiency and increase the cost of production in broilers. Therefore, it is an important task for poultry breeders to breed broilers with low abdominal fat. Abdominal fat deposition is a highly complex biological process, and its molecular basis remains elusive. In this study, we performed transcriptome analysis to compare gene expression profiles at different stages of abdominal fat deposition to identify the key genes and pathways involved in abdominal fat accumulation. We found that abdominal fat weight (AFW) increased gradually from day 35 (D35) to 91 (D91), and then decreased at day 119 (D119). Accordingly, after detecting differentially expressed genes (DEGs) by comparing gene expression profiles at D35 vs. D63 and D35 vs. D91, and identifying gene modules associated with fat deposition by weighted gene co-expression network analysis (WGCNA), we performed intersection analysis of the detected DEGs and WGCNA gene modules and identified 394 and 435 intersecting genes, respectively. The results of the Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that the steroid hormone biosynthesis and insulin signaling pathways were co-enriched in all intersecting genes, steroid hormones have been shown that regulated insulin signaling pathway, indicating the importance of the steroid hormone biosynthesis pathway in the development of broiler abdominal fat. We then identified 6 hub genes (ACTB, SOX9, RHOBTB2, PDLIM3, NEDD9, and DOCK4) related to abdominal fat deposition. Further analysis also revealed that there were direct interactions between 6 hub genes. SOX9 has been shown to bind to proteins required for steroid hormone receptor binding, and RHOBTB2 indirectly regulates the steroid hormones biosynthesis through cyclin factor, and ultimately affect fat deposition. Our results suggest that the genes RHOBTB2 and SOX9 play an important role in fat deposition in broilers, by regulating steroid hormone synthesis. These findings provide new targets and directions for further studies on the mechanisms of fat deposition in chicken.

Commensal microbiota regulate renal gene expression in a sex-specific manner.

The gut microbiome impacts host gene expression not only in the colon but also at distal sites including the liver, white adipose tissue, and spleen. The gut microbiome also influences the kidney and is associated with renal diseases and pathologies; however, a role for the gut microbiome to modulate renal gene expression has not been examined. To determine if microbes modulate renal gene expression, we used whole organ RNA sequencing to compare gene expression in C57Bl/6 mice that were germ free (lacking gut microbiota) versus conventionalized (gut microbiota reintroduced using an oral gavage of a fecal slurry composed of mixed stool). 16S sequencing showed that male and female mice were similarly conventionalized, although Verrucomicrobia was higher in male mice. We found that renal gene expression was differentially regulated in the presence vs. absence of microbiota and that these changes were largely sex specific. Although microbes also influenced gene expression in the liver and large intestine, most differentially expressed genes (DEGs) in the kidney were not similarly regulated in the liver or large intestine. This demonstrates that the influence of the gut microbiota on gene expression is tissue specific. However, a minority of genes (n = 4 in males and n = 6 in females) were similarly regulated in all three tissues examined, including genes associated with circadian rhythm (period 1 in males and period 2 in females) and metal binding (metallothionein 1 and metallothionein 2 in both males and females). Finally, using a previously published single-cell RNA-sequencing dataset, we assigned a subset of DEGs to specific kidney cell types, revealing clustering of DEGs by cell type and/or sex.NEW & NOTEWORTHY It is unknown whether the microbiome influences host gene expression in the kidney. Here, we utilized an unbiased, bulk RNA-sequencing approach to compare gene expression in the kidneys of male and female mice with or without gut microbiota. This report demonstrates that renal gene expression is modulated by the microbiome in a sex- and tissue-specific manner.

Single-cell RNA sequencing reveals XBP1-SLC38A2 axis as a metabolic regulator in cytotoxic T lymphocytes in multiple myeloma

The mechanisms underlying the functional impairment and metabolic reprogramming of T lymphocytes in multiple myeloma (MM) have not been fully elucidated. In this study, single-cell RNA sequencing was used to compare gene expression profiles in T cells in bone marrow and peripheral blood of 10 newly diagnosed MM patients versus 3 healthy donors. Unbiased bioinformatics analysis revealed 9 cytotoxic T cell clusters. All 9 clusters in MM had higher expression of senescence markers (e.g., KLRG1 and CTSW) than the healthy control; some had higher expression of exhaustion-related markers (e.g., LAG3 and TNFRSF14). Pathway enrichment analyses showed downregulated amino acid metabolism and upregulated unfolded protein response (UPR) pathways, along with absent expression of glutamine transporter SLC38A2 and increased expression of UPR hallmark XBP1 in cytotoxic T cells in MM. In vitro studies revealed that XBP1 inhibited SLC38A2 by directly binding to its promoter, and silencing SLC38A2 resulted in decreased glutamine uptake and immune dysfunction of T cells. This study provided a landscape description of the immunosuppressive and metabolic features in T lymphocytes in MM, and suggested an important role of XBP1-SLC38A2 axis in T cell function.

Transcriptome Discovery of Genes in the Three Phases of Autophagy That Are Upregulated During Atrial Fibrillation.

Background: Autophagy may contribute to the maintenance of atrial fibrillation (AF), but no previous study has concurrently surveyed all 3 phases of autophagy, namely autophagosome formation, lysosome formation, and autophagosome-lysosome fusion. Here we aimed to identify disorders involving various phases of autophagy during AF. Methods and Results: We used bioinformatic techniques to analyze publicly available DNA microarray datasets from the left atrium (LA) and right atrium (RA) of 7 patients with AF and 6 patients with normal sinus rhythm who underwent valvular surgeries. We compared gene expression levels in the LA (AF-LA) and RA of patients with AF with those in the LA and RA of patients with normal sinus rhythm. Several differentially expressed genes in the AF-LA sample were significantly associated with the Gene Ontogeny term 'Autophagy', indicating that the expression of autophagic genes was specifically altered in this dataset. In particular, the expression of genes known or suspected to be involved in autophagosome formation (autophagy related 5 [ATG5], autophagy related 10 [ATG10], autophagy related 12 [ATG12], and light chain 3B [LC3B]), lysosome formation (lysosomal associated membrane protein 1 [LAMP1] and lysosomal associated membrane protein 2 [LAMP2]), and autophagosome-lysosome fusion (synaptosome associated protein 29 [SNAP29], SNAP associated protein [SNAPIN], and syntaxin 17 [STX17]) was significantly upregulated in the LA-AF dataset. Conclusions: Autophagy is activated excessively in, and may perpetuate, AF.

Aquaporin-4 as a New Potential Molecular Biomarker for Prognosis of Low-Grade Glioma: Comprehensive Analysis Based on Online Platforms

Aquaporin-4 (AQP4) is a significant factor in transcellular and transepithelial water movement, and abnormal expression of AQP4 has been detected in many types of tumors. The purpose of this study was to explore its role in low-grade gliomas (LGG) using freely available online bioinformatics tools. OncoLnc database was used to analyze Cox coefficients and compare AQP4 expression between various types of tumors; Tumor Immune Estimation Resource database and Gene Expression Profiling Interactive Analysis were used to compare gene expression between LGG and normal tissues; University of California Santa Cruz Xena browser generated Kaplan-Meier survival curves in the LGG cohort in The Cancer Genome Atlas and subgroups; LinkedOmics database screened the most relevant genes based on Pearson correlation coefficient; Gene Ontology Biological Process and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed through gene set enrichment analysis to explore possible molecular mechanisms. LGG had higher AQP4 expression compared with normal tissues and ranked first among 21 different types of cancer (P < 0.05). The oligodendroglioma group had the lowest AQP4 expression and the longest overall survival (OS) (P < 0.05). LGG with astrocytoma, isocitrate dehydrogenase mutation, or 1p/19q codeletion had lower AQP4 expression and longer OS (P < 0.001). LGG with lower AQP4 expression, without 1p/19q codeletion, without chemotherapy, and with or without radiation therapy had longer OS (P < 0.05). AQP4 and coexpressed genes were involved in complex biological processes in LGG, including regulation of neurotransmitter level, peroxisome proliferator-activated receptor signaling pathway, cell adhesion molecules, and others. AQP4 is a prognostic marker in LGG and its subgroups. Patients with lower AQP4 expression may have longer OS.

Comparative transcriptomic analysis of germinating rice seedlings to individual and combined anaerobic and cold stress

BackgroundRice is one of the most important cereals consumed worldwide. Two major abiotic factors affecting rice plants in different growth stages are flooding stress and cold stress. These abiotic stresses can take place independently or simultaneously and significantly affect rice plants during germination and seedling growth. Fortunately, a wide array of phenotypic responses conferring flooding stress and chilling stress tolerance exist within the rice germplasm, indicating the presence of different molecular mechanisms underlying tolerance to these stresses. Understanding these differences may assist in developing improved rice cultivars having higher tolerance to both stresses. In this study, we conducted a comparative global gene expression analysis of two rice genotypes with contrasting phenotypes under cold stress, anaerobic stress, and combined cold and anaerobic stress during germination.ResultsThe differential gene expression analysis revealed that 5571 differentially expressed genes (DEGs), 7206 DEGs, and 13279 DEGs were identified under anaerobic stress, cold stress, and combined stress, respectively. Genes involved in the carbohydrate metabolic process, glucosyltransferase activity, regulation of nitrogen compound metabolic process, protein metabolic process, lipid metabolic process, cellular nitrogen compound biosynthetic process, lipid biosynthetic process, and a microtubule-based process were enriched across all stresses. Notably, the common Gene Ontology (GO) analysis identified three hub genes, namely Os08g0176800 (similar to mRNA-associated protein mrnp 41), Os11g0454200 (dehydrin), and OS10g0505900 (expressed protein).ConclusionA large number of differentially expressed genes were identified under anaerobic, cold conditions during germination and the combination of the two stress conditions in rice. These results will assist in the identification of promising candidate genes for possible manipulation toward rice crops that are more tolerant under flooding and cold during germination, both independently and concurrently.

Differentially expressed genes associated with body size changes and transposable element insertions between Caenorhabditis elegans and its sister species, C. inopinata.

Why the recently discovered nematode Caenorhabditis inopinata differs so greatly from its sibling species Caenorhabditis elegans remains unknown. A previous study showed that C. inopinata has more transposable elements (TEs), sequences that replicate and move autonomously throughout the genome, potentially altering the expression of neighboring genes. In this study, we focused on how the body size of this species has evolved and whether TEs could affect the expression of genes related to species-specific traits such as body size. First, we compared gene expression levels between C. inopinata and C. elegans in the L4 larval and young adult stages-when growth rates differ most prominently between these species-to identify candidate genes contributing to their differences. The results showed that the expression levels of collagen genes were consistently higher in C. inopinata than in C. elegans and that some genes related to cell size were differentially expressed between the species. Then, we examined whether genes with TE insertions are differentially expressed between species. Indeed, the genes featuring C. inopinata-specific TE insertions had higher expression levels in C. inopinata than in C. elegans. These upregulated genes included those related to body size, suggesting that these genes could be candidates for artificial TE insertion to examine the role of TEs in the body size evolution of C. inopinata.

Effect of RNA preservation methods on RNA quantity and quality of field-collected avian whole blood

The transcriptome comprises all RNA molecules in a sample, tissue, or organism. A limitation of comparative transcriptomic studies which compare gene expression between individuals often under some differing exposure or treatment, of wild avian populations continues to be sample preservation of high-quality RNA (i.e., ribonucleic acids that transfer, translate, and regulate the genetic code from DNA into proteins). Field sampling of wild bird blood provides challenges as RNA degradation progresses quickly, due in large part to the high nuclease content of avian blood and because cryopreservation is often not feasible at remote locations. The introduction of commercial buffers for preservation of RNA enables field-collected studies as these buffers deactivate nucleases which degrade target nucleic acids. We seek to compare the effectiveness of widely available RNA stabilizing buffers, RNAlater (Ambion) and DNA/RNA Shield (Zymo) at varying concentrations along with a dry ice-based flash freezing method to determine optimal preservation methods for field-collected avian blood samples. To determine optimal preservation methods, we assessed RNA quantity and quality metrics: RNA integrity numbers (RINe), rRNA ratios, and total RNA concentration. Nucleated red blood cells, a characteristic common across non-mammalian vertebrates, provide sufficient transcriptionally active material enabling the identification of potentially active gene regions from small and non-lethal samples (∼ 20 μl). A protocol was also optimized for total RNA extraction from avian blood samples with small starting volumes enabling sampling of birds with a minimum threshold of 5 g body mass. We found that RNA preservation buffers, RNAlater and DNA/RNA Shield at all concentrations provide sample protection from RNA degradation. We recommend that caution be exercised when using dry ice-based flash-freezing alone for sample preservation as these samples resulted in lower quality measures then samples in preservation buffer. Total RNA concentration was generally not affected by preservation treatment and may vary due to differences in initial sample volumes.

How larvae and life cycles evolve

Marine larvae have factored heavily in pursuits to understand the origin and evolution of animal life cycles. Recent comparisons of gene expression and chromatin state in different species of sea urchin and annelid show how evolutionary changes in embryonic gene regulation can lead to markedly different larval forms.

Abstract 1684: Plasminogen activator inhibitor-1 (PAI-1) promotes cisplatin resistance in ovarian cancer cells by inhibiting ROS-mediated apoptosis and ferroptosis

Abstract Resistance to platinum-based chemotherapy is one of major causes of therapy failure and tumor recurrence in ovarian cancer patients. However, the molecular mechanism of platinum resistance is not fully understood. Here, we compared gene expression between cisplatin-sensitive and -resistant ovarian cancer cells using gene expression microarray analysis and found the significantly up-regulated expression of SERPINE1 encoding plasminogen activator inhibitor-1 (PAI-1), a serine protease regulating the fibrinolysis and extracelluar matrix, in cisplatin-resistant ovarian cancer cells. The correlation of high PAI-1 expression with low cisplatin sensitivity was confirmed in ovarian cancer cells. PAI-1 overexpression in ovarian cancer cells significantly decreased the sensitivity to cisplatin, whereas PAI-1 down-regulation by SERPINE1 siRNA or PAI-1 inhibitor increased cisplatin sensitivity. PAI-1 inhibited cisplatin-induced apoptosis in ovarian cancer cells by suppressing reactive oxygen species (ROS) production and DNA damage. PAI-1 also decreased cisplatin-induced ferroptosis through the upregulation of GPX4 levels. Furthermore, Kaplan-Meier survival analysis using TGGA data demonstrated that high PAI-1 expression was significantly associated with shorter platinum-free survival of patients with ovarian cancer. Collectively, these results illustrate that PAI-1 promotes cisplatin resistance in ovarian cancer cells by inhibiting ROS-mediated apoptosis and ferroptosis, suggesting that targeting PAI-1 increases the sensitivity to cisplatin in ovarian cancer. Citation Format: Mi Jeong Kwon, Ha Yeong Chae, So Young Lee, Shin Ung Kang, Tae Woong Jung, Soo Youn Cho, Jinil Han, Hyojin Jeong, Chang Ohk Sung, Young Kee Shin. Plasminogen activator inhibitor-1 (PAI-1) promotes cisplatin resistance in ovarian cancer cells by inhibiting ROS-mediated apoptosis and ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1684.

Abstract 2580: Nuclear receptor coactivator 3 (NCOA3) fuels oncogenic pathways in uveal melanoma

Abstract Background: Uveal melanoma (UM) originates from melanocytes residing in the uveal tract of an eye. Despite effective local control, approximately half of patients develop metastatic disease resistant to systemic chemotherapy and presenting poor response to immunotherapy or targeted therapy. UM patients have a median overall survival of 6 to 12 months. The basis of the pathogenesis of UM are mutations in GNAQ and GNA11 genes resulting in constitutive activation of PKC/MAPK signaling. Here, we analyzed the role of NCOA3, a transcription factor coactivator that was previously shown to be modified by PKC in multiple cancer, as a factor potentiating oncogenic outcome of multiple oncogenic pathways activated in UM downstream of GNAQ or GNA11. Methods: We examined the impact of altered NCOA3 mRNA in UM, using publicly available TCGA dataset. Next, we used CRISPR/Cas9 technology to delete NCOA3 protein in MP41 UM cell line. We evaluated proliferation of MP41/NCOA3 KO cell subline and its parental counterpart in vitro by a colony formation assay. Next, we analyzed growth of xenografts of MP41/NCOA3 KO and parental cells in SCID/beige mice. To understand the molecular consequences of NCOA3 activity in UM, we performed a genome wide comparison of gene expression in MP41/NCOA3 KO and parental cells. Functional enrichment analysis was used to identify classes of genes associated with NCOA3 activity and determine similarities with datasets obtained by targeting proteins of GNAQ/GNA11 pathways and inhibiting activity of NCOA3 using a tool small molecule inhibitor SI-2 in UM cell lines. Results: We found that NCOA3 overexpression negatively correlated with UM patients’ progression free and overall survival. Knockout of NCOA3 gene in MP41 UM cells substantially reduced their proliferation in vitro and xenograft growth in mice. Global gene expression analysis revealed that NCOA3 activity is linked with transcriptional signatures associated with cell mitotic activity (cell cycle and DNA replication) and transcriptional machinery binding (especially affecting E2F and myc regulated programs). The MP41 NCOA3 KO normalized enrichment scores (NES) show high concordance with NES obtained after silencing GNAQ or GNA11 in UM cell lines and pharmacological targeting PKC or NCOA3. Conclusions: Inhibition of NCOA3 activity inhibits several oncogenic pathways in a concerted manner, limiting UM cell survival and tumor growth. Our results deliver proof of concept that NCOA3 is a master transcriptional co-regulator of oncogenic signaling in uveal melanoma and a potential therapeutic target. Citation Format: Aleksandra Rusin, Maria E. Ruiz Echartea, Darlene G. Skapura, Karen Berman deRuiz, Christel M. Davis, Eric A. Ehli, Sandra L. Grimm, Christian Coarfa, Salma Kaochar. Nuclear receptor coactivator 3 (NCOA3) fuels oncogenic pathways in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2580.

Abstract 3370: Liquid biopsy approaches to determine tumor cell heterogeneity in advanced prostate cancer

Abstract Aggressive variants of prostate cancer (AVPC) emerge from prostate adenocarcinoma following enduring therapeutic pressure. They grow independent of the androgen receptor, progress rapidly and can show traits of neuroendocrine (NE) differentiation. As progression to AVPC is not well detected by prostate specific antigen monitoring, new biomarkers are urgently needed. The aim of this study is to evaluate the detection of circulating tumor cells (CTCs) in combination with gene expression analysis of NE genes to identify AVPC patients with neuroendocrine traits. Blood samples were collected from patients suffering from AVPC (n=78, including 25 patients with histological evidence of NEPC) as well as from patients with hormone-sensitive prostate cancer (HSPC) as controls (n=12) at the University Medical Center Hamburg-Eppendorf. CTC counts were determined by CellSearch analysis. In parallel, CTCs were immuno-magnetically enriched using the AdnaTest and bulk gene expression of prostate-specific and NE genes was analyzed by semi-quantitative PCR. From a subset of patients, CTCs were additionally enriched by Parsortix to compare gene expression of CTCs with EpCAM-based or size-based CTC enrichment. Using CellSearch analysis, CTCs were found in 88.9 % of AVPC patients. CTC counts ranged between 0 – 20,000 CTCs/7.5 ml of blood with a median of 32 CTCs/7.5 ml. Similarly, 81.8 % of NEPC patients were positive for CTCs with a median count of 35 CTCs/7.5 ml (range 0-13,000). In contrast, CTCs were found in only 50 % of HSPC patients at lower concentrations (median count of 0.5 CTCs/7.5 ml, range 0-34). CTC counts were significantly elevated in AVPC and NEPC patients compared to HSPC (p = 0.002, p = 0.012), but no significant differences were found between AVPC and NEPC samples.In 93.8 % of AVPC samples, CTCs expressed prostate-specific genes such as KLK3 or PSMA. At least one of the NE transcripts was detected in 37.5 % of AVPC patients. In CTCs from NEPC patients, the positivity for prostate markers was significantly reduced to 66.6 % (p = 0.01) and the positivity for NE markers was increased to 61.1 %. A random Forrest model trained on all analyzed transcripts allowed for a distinction of HSPC and NEPC with an AUC of 79.5 %. Comparison of CTC enrichment strategies in a subset of 13 patients showed similar NE marker detection by size-based enrichment (53.8 % NE-positive) compared to the AdnaTest (61.5 % NE-positive). AVPC and NEPC patients show a high CTC burden that will allow subsequent molecular analyses, and CTC counts allowed a distinction between blood samples from HSPC and NEPC patients. Gene expression analysis revealed a high degree of inter-patient heterogeneity for neuroendocrine-specific transcripts with a reduction of prostate markers in NEPC patients. Future molecular characterization and longitudinal monitoring of CTCs will shed more light on the evolution of AVPC and NEPC. Citation Format: Lina Merkens, Sarah Greimeier, Sabine Riethdorf, Klaus Pantel, Gunhild von Amsberg, Stefan Werner. Liquid biopsy approaches to determine tumor cell heterogeneity in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3370.

Abstract 674: CRISPR and drug screens identify ERK as the mediator of IFNg-induced melanoma growth inhibition

Abstract Interferon-gamma (IFNg) is critical for the success of immune checkpoint blockade therapy. Disruption of IFNg signaling makes tumors resistant to anti-PD-1/L1 treatment primarily by evasion of the growth inhibition (GI) induced by this cytokine. However, the pathways mediating IFNg-induced GI are largely unknown. We used two independent approaches to delineate the IFNg GI pathway, a chemical genomics screen and a whole genome targeting CRISPR/Cas9 screen in an IFNg GI-sensitive patient-derived melanoma (PDM) cell line. Drug screening revealed that treatment with RAF and ERK inhibitors disrupted IFNg GI in PDM cells. For the CRISPR screen, gene-level analysis showed that the magnitude of enrichment for guide RNAs targeting ERK2 in IFNg-treated cells was comparable to proximal IFNg signaling proteins like JAK1, STAT1, IFNGR2, and JAK2 (&amp;gt;26-fold enrichment for ERK2, p = 2.28e-07). Thus, results from both these experiments converged upon ERK as the primary downstream target of IFNg essential for GI induction. We validated the involvement of ERK by detecting a sustained increase in phospho-ERK1/2 levels following IFNg treatment. In live imaging experiments, we found that blocking ERK activity with the ERK inhibitor Ulixertinib blocks the induction of cell death after IFNg treatment in 17 of 23 (~74%) IFNg-sensitive PDM lines covering all the MAPK mutant and triple wildtype molecular subtypes of melanoma. Decreasing and increasing ERK activity levels using shRNA and overexpression approaches also led to a decrease and increase in IFNg-mediated GI, respectively. We have identified NOXA and DR5 as likely mediators of cell death through the IFNg-ERK GI pathway by comparing gene expression profiles of control and IFNg-treated PDM cells. Finally, phorbol 12-myristate 13-acetate (PMA), a known ERK activator, synergizes with IFNg treatment to induce increased GI in PDM cells (Bliss synergy score 16.87, p = 1.83e-123). In summary, the IFNg-ERK signaling axis mediates cell death downstream of IFNg treatment in melanoma cells. This pathway is active in all melanoma subtypes, making it an attractive target to enhance IFNg GI in tumor cells. Our results provide a new understanding of the IFNg GI pathway that will also be crucial to define mechanisms of GI resistance in tumor cells. Citation Format: Ameya S. Champhekar, Rachel Heymans, Cynthia Gonzalez, Guillem Turon Font, Daniel Karin, June Lee, Robert Damoiseaux, Antoni Ribas. CRISPR and drug screens identify ERK as the mediator of IFNg-induced melanoma growth inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 674.