Multiple Gene Sets Research Articles

5-hydroxymethylcytosine sequencing in plasma cell-free DNA identifies unique epigenomic features in prostate cancer patients resistant to androgen deprivation therapies.

Currently, no biomarkers are available to identify resistance to androgen-deprivation therapies (ADT) in men with hormone-naive prostate cancer. Since 5-hydroxymethylcytosines (5hmC) in gene body are associated with gene activation, in this study, we evaluated whether 5hmC signatures in cell-free DNA (cfDNA) predicts early resistance to ADT. We collected a total of 139 serial plasma samples from 55 prostate cancer patients receiving ADT at three time points including baseline (prior to initiating ADT, N=55), 3-month (after initiating ADT, N=55), and disease progression (N=15) within 24 months or 24-month if no progression was detected (N=14). To quantify 5hmC abundance across the genome, we used selective chemical labeling sequencing and mapped sequence reads to individual genes. Differential methylation analysis in baseline samples identified significant 5hmC difference in 1,642 of 23,433 genes between patients with and without progression (false discovery rate, FDR<0.1). Patients with disease progression showed significant 5hmC enrichments in multiple hallmark gene sets with androgen responses as top enriched gene set (FDR=1.19E-13). Interestingly, this enrichment was driven by a subgroup of patients featuring a significant 5hmC hypermethylation in the gene sets involving AR , FOXA1 and GRHL2 . To quantify overall activities of these gene sets, we developed a gene set activity scoring algorithm and observed significant association of high activity scores with poor progression-free survival (P<0.05). Longitudinal analysis showed that the high activity scores were significantly reduced after 3-months of initiating ADT (P<0.0001) but returned to higher levels when the disease was progressed (P<0.05). This study demonstrates that 5hmC-based activity scores from gene sets involved in AR , FOXA1 and GRHL2 may be used as biomarkers to determine early treatment resistance, monitor disease progression, and potentially identify patients who would benefit from upfront treatment intensification.

Drinking motives and alcohol sensitivity mediate multi-dimensional genetic influences on alcohol use behaviors.

Genetic influences account for a substantial proportion of individual differences in alcohol use behaviors (AUBs). However, multiple distinct sets of genes are linked to different AUBs, which may explain their dramatic variability in risk factors and manifestations. In this study, we explore whether intermediate neurobiological traits and alcohol-related cognitions mediate the relationship between polygenic scores (PGS) and multiple AUBs, with the aim to better understand processes captured by different genetic profiles. Using results from prior genome-wide association studies, we derived PGS for 6 AUBs in participants from Spit for Science, a longitudinal study of college students in the U.S. (n=4,549). Self-report measures included personality traits, alcohol expectancies, drinking motivations, and alcohol sensitivity measures as well as drinking frequency, drinking quantity, alcohol use disorder (AUD) symptoms, and maximum drinks in 24 hours. Using linear regression and multiple mediation models, we investigated the direct and indirect effects of PGS on AUBs. In univariable regression results, PGSs indexing broad AUB dimensions such as drinks per week (DPW) and AUD predicted higher levels of sensation-seeking and multiple drinking motives, while BeerPref PGSs (indexing a variable pattern of alcohol problems associated with a preference for beer) predicted higher negative urgency and lower alcohol sensitivity. Mediational models indicated strong direct and indirect effects of DPW PGSs on multiple AUBs via social/enhancement drinking motives and alcohol sensitivity, indirect effects of AUD PGSs on AUD symptoms via coping motives, and indirect effects of BeerPref PGS on all AUBs via the joint effect of mediators including alcohol sensitivity. These findings provide initial evidence that the genetic influences on different AUBs are associated with and partially mediated by intermediate neurobiological and cognitive factors, which may be more amenable to intervention. Greater focus on drinking motives and alcohol sensitivity is warranted in genetic research, as well as attention to the heterogeneous pathways linking genes to alcohol use outcomes.

High-fidelity CRISPR/Cas12a dual-crRNA screening reveals novel synergistic interactions in hepatocellular carcinoma.

: CRISPR/Cas12a-based combinational screening has shown remarkable potential for identifying genetic interactions. Here, we describe an innovative method for combinational genetic screening with rapid construction of a dual-CRISPR RNA (crRNA) library using gene splicing through overlap extension PCR (SOE PCR) and the adoption of CeCas12a, which we previously identified with strict PAM recognition and low off-targeting to guarantee fidelity and efficiency. The custom-pooled SOE crRNA array (SOCA) library for double-knockout screening could be conveniently constructed in the laboratory for widespread use, and the CeCas12a-mediated high-fidelity screen displayed good performance even under a negative selection screen. By designing a SOCA dual-crRNA library that covered most of the kinase and metabolism-associated gene targets of FDA-approved drugs implicated in hepatocellular carcinoma (HCC) tumourigenesis, novel cross-talk between the two gene sets was negatively selected to inhibit HCC cell growth in vitro and in vivo and was validated using virtual double-knockdown screening based on TCGA databases. Thus, this rapid, efficient and high-fidelity double-knockout screening system is promising for systemically identifying potential genetic interactions between multiple gene sets or combinations of FDA- approved drugs for clinical translational medicine in the future.

Using de novo transcriptomes to decipher the relationships in cutthroat trout subspecies (Oncorhynchus clarkii).

For almost 200 years, the taxonomy of cutthroat trout (Oncorhynchus clarkii), a salmonid native to Western North America, has been in flux as ichthyologists and fisheries biologists have tried to describe the diversity within these fishes. Starting in the 1950s, Robert Behnke reexamined the cutthroat trout and identified 14 subspecies based on morphological traits, Pleistocene events, and modern geographic ranges. His designations became instrumental in recognizing and preserving the remaining diversity of cutthroat trout. Over time, molecular techniques (i.e. karyotypes, allozymes, mitochondrial DNA, SNPs, and microsatellite arrays) have largely reinforced Behnke's phylogenies, but have also revealed that some relationships are consistently weakly supported. To further resolve these relationships, we generated de novo transcriptomes for nine cutthroat subspecies, as well as a Bear River Bonneville form and two Colorado River lineages (blue and green). We present phylogenies of these subspecies generated from multiple sets of orthologous genes extracted from our transcriptomes. We confirm many of the relationships identified in previous morphological and molecular studies, as well as discuss the importance of significant differences apparent in our phylogenies from these studies within a geological perspective. Specific findings include three distinct clades: (1) Bear River Bonneville form and Yellowstone cutthroat trout; (2) Bonneville cutthroat trout (n = 2); and (3) Greenback and Rio Grande cutthroat trout. We also identify potential gene transfer between Bonneville cutthroat trout and a population of Colorado River green lineage cutthroat trout. Using these findings, it appears that additional groups warrant species-level consideration if other recent species elevations are retained.

Abstract PO2-09-05: Building genomic and transcriptomic data among African American and Black patients with triple-negative inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive breast cancer associated with poor response and early metastases. It represents a health disparity, with higher incidence and worse outcomes in Black compared to White patients. Further, IBC is enriched for the more aggressive triple-negative receptor subtype, TN-IBC, and this subtype is more prevalent among Black IBC patients. It is critical to identify genetic and molecular features that may guide better treatments for these aggressive cancers across races, and thus imperative to identify and increase racial representation among bioinformatics datasets. We previously performed comprehensive genomic and transcriptomic analyses for TN-IBC patient samples and here examine the differences among self-identified African American or Black (AA/B) and other races among IBC patients. Methods: As previously described, we collected matched blood and baseline tumor samples before treatment from 19 patients with primary TN-IBC. We performed whole exome and RNA sequencing (RNA-Seq) on these samples and compared mutations and differentially expressed genes (DEGs). Results were compared between 3 AA/B patient tissues vs the remaining 16 (Other) cases. Results: Mutations shared by all AA/B patients included amplification of the five most commonly mutated hallmark genes in the complete cohort, ARNT, BCL9, DDR2, FCGR2B and LMNA (all 100% in AA/B patients versus all 50% in the Other cohort). All AA/B patients had a Notch1 mutation (two deletions and one missense mutation, 100% AA/B vs 6% Other (one deletion)). Comparing differentially expressed genes and gene set enrichment analyses of the Broad molecular signatures database (MSigDB) demonstrates the majority of DEGs are downregulated in these AA/B patients. Six of the top 20 downregulated gene sets from the MSigDB C2 group relate to breast cancer subtype and among this TNIBC cohort AA/B tumor expression was downregulated for luminal and normal subtype related genes in multiple gene sets and enriched in the Lien metaplastic related genes. In the MSigDB C5 sets, over half of the top 20 enriched gene ontology sets among AA/B patients related to chromosomes, chromatin, spindle assembly or histone binding, while 16 of the top 20 downregulated enriched sets related to immune function. Multiple KEGG pathways enriched in AA/B cases related to RNA processing and expression and DNA replication and repair. Lastly, hallmark gene sets enriched in AA/B patients were cell cycle related, and targets of Myc, Hedgehog, B-catenin, TGFb, and TNFa via NFKb, while top significantly downregulated sets related to metabolism and immune response. Conclusion: While findings from a small sample size can only be considered hypothesis generating, some patterns emerge from the data among this rare cohort of AA/B TN IBC patients. These tumors appear to be devoid of luminal signals, characterized by cell cycle and chromatin remodeling signals, enriched for signaling typical of stem cell self-renewal, and perhaps more immune-deprived than other TN-IBC. Additional work to build racially diverse IBC transcriptomic data are needed to develop strategies to understand and improve outcomes for these patients. Citation Format: Wencai Ma, Li Zhao, Gayathri Devi, Savitri Krishnamurthy, Larry Coffer, Angela Alexander, Megumi Kai, Xiaoping Wang, Huong Le-Petross, Miral Patel, Bisrat Debeb, Chandra Bartholomeusz, Jianhua Zhang, Xingzhi Song, Andrew Futreal, Naoto Ueno, Rachel Layman, Anthony Lucci, Jing Wang, Wendy Woodward. Building genomic and transcriptomic data among African American and Black patients with triple-negative inflammatory breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-09-05.

Abstract PO1-24-12: Investigating the immunological function of alpha-2-glycoprotein 1, zinc-binding in regulating tumor response in the breast cancer microenvironment

Abstract Purpose: Tumor immunology has attracted considerable attention as an innovative therapeutic strategy for various types of cancer. Elucidating the unique immunoregulatory mechanisms in the breast cancer microenvironment will assist in the development of novel treatment strategies. Recently, we and other researchers have found that androgen receptor (AR) expression is associated with the immunosuppressive phenotype in the breast cancer microenvironment, suggesting some immunoregulatory function in breast cancer; however, the mechanism remains unclear. Here, we focused on AR-dependent secreted protein, alpha-2-glycoprotein 1, zinc-binding (ZAG) encoded by the AZGP1 gene in breast cancer, which is structurally similar to HLA class I and is implicated in immune regulation. In this study, we investigated the immunological function of AZGP1/ZAG in the breast cancer microenvironment. Methods: We performed a gene set enrichment analysis (GSEA) to screen the biological processes associated with AZGP1 expression using a gene expression profile dataset of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). Subsequently, we analyzed the correlation between AZGP1 expression and the immune cell composition in breast cancer tissues estimated with the CIBERSORTx using METABRIC and another dataset of The Sweden Cancerome Analysis Network-Breast. In our previous study of 45 breast cancer tissue samples, we evaluated the infiltration of 11 types of immune cells using flow cytometry (FCM). ZAG expression was further evaluated by immunohistochemistry, and the relationship between ZAG expression and the immune cell composition in breast cancer tissue was analyzed. Based on the results of this analysis (shown in the results section) we hypothesized that ZAG has some effect on the macrophage (Mφ). The action of ZAG in M1/M2 polarization models constructed using primary culture of human peripheral blood mononuclear cells (PBMC)-derived Mφ was assessed based on the expression of M1/M2 differentiation markers (CD86, CD80/CD163, MRC1) and HLA class I/II expression evaluated by FCM (n=15 each). Results: GSEA demonstrated that AZGP1 expression was negatively correlated with multiple gene sets representing immunological processes, including inflammatory response, allograft rejection, interferon gamma response, IL6/JAK/STAT3 signaling, complement, and IL2/STAT5 signaling. Analysis by CIBERSORTx showed that AZGP1 expression was negatively correlated with the absolute score (the absolute abundance of total immune cell infiltration), Mφ M1, NK cells activated, CD4+ T memory activated, and CD8+ T (r&amp;lt;-3 and p&amp;lt; 0.05). Analyses of our in-house dataset using breast cancer tissue showed that ZAG expression was associated with decreased infiltration of monocytes/macrophages, non-classical monocytes, and myeloid-derived suppressor cells in breast cancer tissues assessed by FCM. In the in vitro analyses, ZAG decreased the expression of CD80, CD163, MRC1, and HLA classes I and II in the M1 polarization model and the expression of CD163 and MRC1 in the M2 polarization model. Conclusion: AZGP1/ZAG was associated with an immunosuppressive phenotype and reduced infiltration of specific immune cell subsets, particularly Mφ, into breast cancer tissues. In the in vitro analysis, ZAG demonstrated some regulatory effects on the phenotypic change of Mφ. Our findings strongly suggest ZAG is a novel mediator of AR-dependent immunomodulation in the breast cancer microenvironment, laying the foundation for future studies to elucidate the immunological role of ZAG in breast cancer. Citation Format: Toru Hanamura, Kozue Yokoyama, Shigehisa Kitano, Hiroshi Kagamu, Makiko Yamashita, Mayako Terao, Takuho Okamura, Nobue Kumaki, Katsuto Hozumi, Takayuki Iwamoto, Chikako Honda, Sasagu Kurozumi, Jennifer Richer, Naoki Niikura. Investigating the immunological function of alpha-2-glycoprotein 1, zinc-binding in regulating tumor response in the breast cancer microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-12.

Enhanced epithelial-mesenchymal transition signatures are linked with adverse tumor microenvironment, angiogenesis and worse survival in gastric cancer.

Epithelial-mesenchymal transition (EMT) is a crucial mechanism that facilitates cancer cell metastasis. Despite its importance, the clinical significance of EMT in gastric cancer (GC) patients has yet to be clearly demonstrated. For gauging the extent of EMT in GC, we employed gene set variation analysis to score 807 patient samples from two large cohorts: TCGA and GSE84437. In both cohorts, EMT high GC showed a significant association with worse overall survival (hazard ratio (HR) = 1.74, p = 0.011 and HR = 2.01, p < 0.001, respectively). This association was stronger when considering the EMT signature score compared to the individual expressions of EMT-related genes (CDH1, CDH2, VIM, and FN1). While the EMT signature level did not differ among various cancers, high EMT signature specifically correlated with survival in GC alone. Mucinous and diffuse histological types exhibited higher EMT levels compared to others (p < 0.001), and the EMT signature level was correlated with tumor depth and AJCC stage (all p < 0.001). Interestingly, the EMT score was an independent factor for overall and disease-specific survival (multivariate; p = 0.006 and 0.032, respectively). EMT high GC displayed a lower fraction of Th1 cells and a higher fraction of dendritic cells, M1 macrophages and several stromal cells. EMT high GC exhibited an inverse correlation with cell proliferation-related gene sets. While they significantly enriched multiple pro-cancerous gene sets, such as TGF-β signaling, hypoxia, and angiogenesis. The presence of EMT signature in a bulk tumor was linked to TGF-β signaling, hypoxia, and angiogenesis, and was also associated with poorer survival outcomes in GC patients.

Abstract 7044: Transcriptome and TCR repertoire in human telomerase reverse transcriptase peptide vaccine treated metastatic prostate cancer patients

Abstract Human telomerase reverse transcriptase (hTERT) vaccine has found to be effective for most of the participated de novo metastatic prostate cancer patients in clinical studies. Nevertheless, the molecular mechanisms behind the immune response induced by the hTERT vaccine are not fully elucidated. Among the cohort of 21 patients treated with androgen deprivation therapy, radiotherapy and hTERT vaccine, we used RNA-sequencing and TCR-sequencing (20 and 14 pre-treatment and 20 and 21 post-treatment samples, respectively) to inspect the changes in transcriptome and TCR-repertoire after the hTERT vaccine treatment and differences between patients that had early response and patients that had late or no response. We found multiple hallmarks being shifted after the treatment and being different between early and late responders. Also, a change in activity of multiple gene set signatures was detected after the treatment and activity of part of the gene set signatures predicted survival. Deconvolution of cell type composition from RNA-sequencing data revealed changes in immune cell abundances after the treatment. TCR-sequencing revealed changes in composition and diversity of TCR repertoires after the treatment and these differed between early and late responders. The results of this study will help to predict patients’ responses to the cancer vaccine and to further understand the mechanisms behind successful cancer vaccines. Citation Format: Reetta Nätkin, Julia Elisabeth Simensen, Nikolai Engedal, Espen Basmo Ellingsen, Marita Westhrin, Ingunn Westgaard, Eivind Hovig, Wolfgang Lilleby, Matti Nykter, Alfonso Urbanucci. Transcriptome and TCR repertoire in human telomerase reverse transcriptase peptide vaccine treated metastatic prostate cancer patients [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 7044.

Genome-wide transcriptional response to silver stress in extremely halophilic archaeon Haloferax alexandrinus DSM 27206 T

BackgroundThe extremely halophilic archaeon Haloferax (Hfx.) alexandrinus DSM 27206 T was previously documented for the ability to biosynthesize silver nanoparticles while mechanisms underlying its silver tolerance were overlooked. In the current study, we aimed to assess the transcriptional response of this haloarchaeon to varying concentrations of silver, seeking a comprehensive understanding of the molecular determinants underpinning its heavy metal tolerance.ResultsThe growth curves confirmed the capacity of Hfx. alexandrinus to surmount silver stress, while the SEM–EDS analysis illustrated the presence of silver nanoparticles in cultures exposed to 0.5 mM silver nitrate. The RNA-Seq based transcriptomic analysis of Hfx. alexandrinus cells exposed to 0.1, 0.25, and 0.5 mM silver nitrate revealed the differential expression of multiple sets of genes potentially employed in heavy-metal stress response, genes mostly related to metal transporters, basic metabolism, oxidative stress response and cellular motility. The RT-qPCR analysis of selected transcripts was conducted to verify and validate the generated RNA-Seq data.ConclusionsOur results indicated that copA, encoding the copper ATPase, is essential for the survival of Hfx. alexandrinus cells in silver-containing saline media. The silver-exposed cultures underwent several metabolic adjustments that enabled the activation of enzymes involved in the oxidative stress response and impairment of the cellular movement capacity. To our knowledge, this study represents the first comprehensive analysis of gene expression in halophillic archaea facing increased levels of heavy metals.

Sex-dimorphic expression of extracellular matrix genes in mouse bone marrow neutrophils.

The mammalian innate immune system is sex-dimorphic. Neutrophils are the most abundant leukocyte in humans and represent innate immunity's first line of defense. We previously found that primary mouse bone marrow neutrophils show widespread sex-dimorphism throughout life, including at the transcriptional level. Extracellular matrix [ECM]-related terms were observed among the top sex-dimorphic genes. Since the ECM is emerging as an important regulator of innate immune responses, we sought to further investigate the transcriptomic profile of primary mouse bone marrow neutrophils at both the bulk and single-cell level to understand how biological sex may influence ECM component expression in neutrophils throughout life. Here, using curated gene lists of ECM components and unbiased weighted gene co-expression network analysis [WGCNA], we find that multiple ECM-related gene sets show widespread female-bias in expression in primary mouse neutrophils. Since many immune-related diseases (e.g., rheumatoid arthritis) are more prevalent in females, our work may provide insights into the pathogenesis of sex-dimorphic inflammatory diseases.

Novel genome-wide associations for effort valuation and psychopathology in children and adults.

The Research Domain Criteria (RDoC) initiative was established by the US National Institute of Mental Health as a multilevel, disorder-agnostic framework for analysis of human psychopathology through designated domains and constructs, including the "Positive Valence Systems" domain focused on reward-related behavior. This study investigates the reward valuation subconstruct of "effort" and its association with genetic markers, functional neurobiological pathways, and polygenic risk scores for psychopathology in 1215 children aged 6-12 and their parents (n = 1044). All participants completed the effort expenditure for rewards task (EEfRT), which assesses "effort" according to two quantitative measures: hard-task choice and reward sensitivity. Genetic association analyses were undertaken in MAGMA, utilizing EEfRT outcome variables as genome-wide association studies phenotypes to compute SNP and gene-level associations. Genome-wide association analyses found two distinct genetic loci that were significantly associated with measures of reward sensitivity and a separate genetic locus associated with hard task choice. Gene-set enrichment analysis yielded significant associations between "effort" and multiple gene sets involved in reward processing-related pathways, including dopamine receptor signaling, limbic system and forebrain development, and biological response to cocaine. These results serve to establish "effort" as a relevant construct for understanding reward-related behavior at the genetic level and support the RDoC framework for assessing disorder-agnostic psychopathology.

The major TMEM106B dementia risk allele affects TMEM106B protein levels, fibril formation, and myelin lipid homeostasis in the ageing human hippocampus

BackgroundThe risk for dementia increases exponentially from the seventh decade of life. Identifying and understanding the biochemical changes that sensitize the ageing brain to neurodegeneration will provide new opportunities for dementia prevention and treatment. This study aimed to determine how ageing and major genetic risk factors for dementia affect the hippocampal proteome and lipidome of neurologically-normal humans over the age of 65. The hippocampus was chosen as it is highly susceptible to atrophy with ageing and in several neurodegenerative diseases.MethodsMass spectrometry-based proteomic and lipidomic analysis of CA1 hippocampus samples from 74 neurologically normal human donors, aged 66–104, was used in combination with multiple regression models and gene set enrichment analysis to identify age-dependent changes in the proteome and lipidome. ANOVA was used to test the effect of major dementia risk alleles in the TMEM106B and APOE genes on the hippocampal proteome and lipidome, adjusting for age, gender, and post-mortem interval. Fibrillar C-terminal TMEM106B fragments were isolated using sarkosyl fractionation and quantified by immunoblotting.ResultsForty proteins were associated with age at false discovery rate-corrected P < 0.05, including proteins that regulate cell adhesion, the cytoskeleton, amino acid and lipid metabolism, and ribosomal subunits. TMEM106B, a regulator of lysosomal and oligodendrocyte function, was regulated with greatest effect size. The increase in TMEM106B levels with ageing was specific to carriers of the rs1990622-A allele in the TMEM106B gene that increases risk for frontotemporal dementia, Alzheimer’s disease, Parkinson’s disease, and hippocampal sclerosis with ageing. Rs1990622-A was also associated with higher TMEM106B fibril content. Hippocampal lipids were not significantly affected by APOE genotype, however levels of myelin-enriched sulfatides and hexosylceramides were significantly lower, and polyunsaturated phospholipids were higher, in rs1990622-A carriers after controlling for APOE genotype.ConclusionsOur study demonstrates that TMEM106B protein abundance is increased with brain ageing in humans, establishes that dementia risk allele rs1990622-A predisposes to TMEM106B fibril formation in the hippocampus, and provides the first evidence that rs1990622-A affects brain lipid homeostasis, particularly myelin lipids. Our data suggests that TMEM106B is one of a growing list of major dementia risk genes that affect glial lipid metabolism.

Associations between prediagnostic aspirin use and ovarian tumor gene expression.

Aspirin use has been associated with reduced ovarian cancer risk, yet the underlying biological mechanisms are not fully understood. To gain mechanistic insights, we assessed the association between prediagnosis low and regular-dose aspirin use and gene expression profiles in ovarian tumors. RNA sequencing was performed on high-grade serous, poorly differentiated, and high-grade endometrioid ovarian cancer tumors from the Nurses' Health Study (NHS), NHSII, and New England Case-Control Study (n = 92 cases for low, 153 cases for regular-dose aspirin). Linear regression identified differentially expressed genes associated with aspirin use, adjusted for birth decade and cohort. False discovery rates (FDR) were used to account for multiple testing and gene set enrichment analysis was used to identify biological pathways. No individual genes were significantly differentially expressed in ovarian tumors in low or regular-dose aspirin users accounting for multiple comparisons. However, current versus never use of low-dose aspirin was associated with upregulation of immune pathways (e.g., allograft rejection, FDR = 5.8 × 10-10 ; interferon-gamma response, FDR = 2.0 × 10-4 ) and downregulation of estrogen response pathways (e.g., estrogen response late, FDR = 4.9 × 10-8 ). Ovarian tumors from current regular aspirin users versus never users were also associated with upregulation in interferon pathways (FDR <1.5 × 10-4 ) and downregulation of multiple extracellular matrix (ECM) architecture pathways (e.g., ECM organization, 4.7 × 10-8 ). Our results suggest low and regular-dose aspirin may impair ovarian tumorigenesis in part via enhancing adaptive immune response and decreasing metastatic potential supporting the likely differential effects on ovarian carcinogenesis and progression by dose of aspirin.

Profiling of miRNAs and their interfering targets in peripheral blood mononuclear cells from patients with chronic myeloid leukaemia.

MicroRNAs may be implicated in the acquisition of drug resistance in chronic myeloid leukemia as they regulate the expression of not only BCR-ABL1 but also genes associated with the activation of drug transfer proteins or essential signaling pathways. To understand the impact of specifically expressed miRNAs in chronic myeloid leukemia and their target genes, we collected peripheral blood mononuclear cells (PBMC) from patients diagnosed with chronic myeloid leukemia (CML) and healthy donors to determine whole miRNA expression by small RNA sequencing and screened out 31 differentially expressed microRNAs (DE-miRNAs) with high expression. With the utilization of miRNA set enrichment analysis tools, we present here a comprehensive analysis of the relevance of DE-miRNAs to disease and biological function. Furthermore, the literature-based miRNA-target gene database was used to analyze the overall target genes of the DE-miRNAs and to define their associated biological responses. We further integrated DE-miRNA target genes to identify CML miRNA targeted gene signature singscore (CMTGSS) and used gene-set enrichment analysis (GSEA) to analyze the correlation between CMTGSS and Hallmark gene-sets in PBMC samples from clinical CML patients. Finally, the association of CMTGSS stratification with multiple CML cell lineage gene sets was validated in PBMC samples from CML patients using GSEA. Although individual miRNAs have been reported to have varying degrees of impact on CML, overall, our results show that abnormally upregulated miRNAs are associated with apoptosis and aberrantly downregulated miRNAs are associated with cell cycle. The clinical database shows that our defined DE-miRNAs are associated with the prognosis of CML patients. CMTGSS-based stratification analysis presented a tendency for miRNAs to affect cell differentiation in the blood microenvironment. Collectively, this study defined differentially expressed miRNAs by miRNA sequencing from clinical samples and comprehensively analyzed the biological functions of the differential miRNAs in association with the target genes. The analysis of the enrichment of specific myeloid differentiated cells and immune cells also suggests the magnitude and potential targets of differentially expressed miRNAs in the clinical setting. It helps us to make links between the different results obtained from the multi-faceted studies to provide more potential research directions.

3D bioengineered neural tissue generated from patient-derived iPSCs mimics time-dependent phenotypes and transcriptional features of Alzheimer's disease.

Several iPSC-derived three-dimensional (3D) cultures have been generated to model Alzheimer's disease (AD). While some AD-related phenotypes have been identified across these cultures, none of them could recapitulate multiple AD-related hallmarks in one model. To date, the transcriptomic features of these 3D models have not been compared with those of human AD brains. However, these data are crucial to understanding the pertinency of these models for studying AD-related pathomechanisms over time. We developed a 3D bioengineered model of iPSC-derived neural tissue that combines a porous scaffold composed of silk fibroin protein with an intercalated collagen hydrogel to support the growth of neurons and glial cells into complex and functional networks for an extended time, a fundamental requisite for aging studies. Cultures were generated from iPSC lines obtained from two subjects carrying the familial AD (FAD) APP London mutation, two well-studied control lines, and an isogenic control. Cultures were analyzed at 2 and 4.5 months. At both timepoints, an elevated Aβ42/40 ratio was detected in conditioned media from FAD cultures. However, extracellular Aβ42 deposition and enhanced neuronal excitability were observed in FAD culture only at 4.5 months, suggesting that extracellular Aβ deposition may trigger enhanced network activity. Remarkably, neuronal hyperexcitability has been described in AD patients early in the disease. Transcriptomic analysis revealed the deregulation of multiple gene sets in FAD samples. Such alterations were strikingly similar to those observed in human AD brains. These data provide evidence that our patient-derived FAD model develops time-dependent AD-related phenotypes and establishes a temporal relation among them. Furthermore, FAD iPSC-derived cultures recapitulate transcriptomic features of AD patients. Thus, our bioengineered neural tissue represents a unique tool to model AD in vitro over time.

#2999 APABETALONE REDUCES CARDIAC EVENTS IN CKD PATIENTS BY DOWNREGULATING FIBROTIC AND INFLAMMATORY PROCESSES

Abstract Background and Aims Major adverse cardiac events (MACE) are a leading cause of mortality in patients with chronic kidney disease (CKD). Apabetalone is an orally available inhibitor of bromodomain &amp; extraterminal (BET) proteins – epigenetic readers that modulate gene expression involved in fibrosis &amp; inflammation. In the phase 3 BETonMACE trial in patients with CVD &amp; diabetes mellitus (NCT02586155), apabetalone reduced MACE by 50% in the subpopulation with CKD (eGFR &amp;lt; 60 mL/min/1.73 m2; HR 0.50 95% CI 0.26,0.96 p = 0.04) indicating favorable response along the kidney-heart axis. This study examines effects of apabetalone in human renal mesangial cells (HRMCs) on fibrotic &amp; inflammatory pathways. In the clinic, we assess plasma levels of fibrotic factors in CKD patients &amp; matched controls after receiving apabetalone. Method TGF-β1 is a pro-fibrotic cytokine that activates HRMCs to over-produce extracellular matrix (ECM). HRMCs were stimulated with TGF-β1 or LPS ± 1-25 µM apabetalone, 0.15-0.5 µM JQ1 or 0.1 µM MZ1 - BET inhibitors (BETi) with chemical scaffolds different than apabetalone. Gene expression was measured by RNA-seq &amp; real-time PCR. RNA-seq was evaluated by Gene Ontology (GO) &amp; Ingenuity Pathway Analysis (IPA). Proteins were measured by immunofluorescence microscopy or ELISA. Collagen gel contraction was assessed in 3D culture. In human subjects, proteomic analysis of plasma was performed via SOMAscan 1.3k 12 hours after 100 mg of apabetalone in CKD patients (n = 8 eGFR &amp;lt; 30 stage 4 or 5) and matched controls (n = 8 eGFR &amp;gt; 60). Results GO analysis of RNA-seq from TGF-β1 stimulated HRMCs showed multiple gene sets associated with ECM production &amp; remodeling in the top 20 affected by BETi. IPA predicted NfkB-RelA and NFkB (complex) were inhibited by apabetalone, consistent with suppressed inflammation. IPA also predicted apabetalone activated pathways of glucose utilization &amp; tolerance of ROS production, including Oxidative Phosphorylation (z-score 5.7, p = 2.95 × 10−3 at 25 µM) &amp; NRF2-Mediated Oxidative Stress Response (z score 2.3, p = 7.9 × 10−12 at 25 µM; z-score 1.6, p = 1.5 × 10−5 at 5 µM). Predicted changes may allow tolerance of elevated glucose. Mechanistically, apabetalone suppressed TGF-β1 induced α-smooth muscle actin (α-SMA) gene expression, a marker of activation, up to 89% (p = 2.8 × 10−6 at 25 µM), and abolished α-SMA filament formation. Consequently, apabetalone opposed TGF-β1 induced collagen gel contraction up to 24% (p = 0.031 at 25 µM). Further, apabetalone countered TGF-β1 induced expression &amp; protein production of key drivers of fibrosis including (a) thrombospondin, an activator of latent TGF-β1 (b) fibronectin, a key ECM component (c) periostin, a promoter of ECM production (d) osteonectin, a regulator of TGF-β1 expression and (e) IL6, a pro-inflammatory cytokine (Table 1). In addition, apabetalone dose dependently opposed LPS stimulated expression of inflammatory genes: IL6 up to 94%, IL1B up to 95% and PTGS2 up to 94% (p&amp;lt;0.001 for each). In all studies, JQ1 or MZ1 had similar activity as apabetalone, confirming on-target BETi effects. In humans, plasma levels of profibrotic and inflammatory markers were reduced 12 hours after receiving apabetalone specifically in those with CKD. Only periostin was reduced in subjects without renal impairment (Table 1). Conclusion Through epigenetic regulation of transcription in HRMCs, apabetalone reduces production of fibrotic &amp; inflammatory factors known to signal along the kidney-heart axis &amp; exacerbate kidney dysfunction. Predicted changes in energy metabolism suggest apabetalone facilitates adaptation to high glucose in kidneys. In a clinical trial, plasma levels of fibrotic &amp; inflammatory factors were reduced specifically in CKD patients receiving apabetalone. Our results provide mechanistic insights into reduced MACE in CKD patients receiving apabetalone in the phase 3 BETonMACE trial &amp; will be further evaluated in an upcoming Phase 3 trial.

Association of enhanced epithelial-mesenchymal transition signature with tumor microenvironment, angiogenesis, and survival in gastric cancer.

e16002 Background: Among the hallmarks of cancer, epithelial-mesenchymal transition (EMT) that epithelial cancer cells gain the characteristics of mesenchymal cells, is one of the key mechanisms of cancer cell metastasis. On the other hand, the clinical relevance of EMT that occurs in gastric cancer patients has not been clearly shown. Methods: To measure the level of EMT in gastric cancer, gene set variation analysis was used to score total of 807 gastric cancer patient samples from two independent large cohorts; TCGA and GSE84437. Results: EMT high gastric cancer was significantly associated with worse overall survival (hazard ratio (HR)=1.74, p=0.011 in the TCGA, and HR=2.01, p&lt;0.001 in the GSE84437), and the association was stronger with EMT signature score compared to expressions of each EMT-related genes, CDH1, CDH2, VIM, or FN1. EMT signature levels were not significantly different among gastric cancer, hepatocellular carcinoma, colorectal cancer, breast cancer, and pancreatic ductal adenocarcinoma, and high level of EMT signature was associated with survival only in gastric cancer. Among the histological types, mucinous and diffuse type had high EMT level compared to tubular, papillary, or signet gastric cancer ( p&lt;0.001). EMT signature level was significantly correlated with tumor depth and AJCC stage (all p&lt;0.001), but not consistently with lymphatic metastasis. Interestingly, the EMT score was independent factor for patient survival including clinicopathological features, not only for overall survival but also disease-specific survival in the TCGA cohort (multivariate; p=0.006 and 0.032, respectively). EMT high gastric cancer was associated with low fraction of Th1 cells and high fraction of dendritic cells and M1 macrophages (all p&lt;0.001 in both cohort), but not with interferon-gamma immune response. EMT high gastric cancer tended to be inversely correlated with cell proliferation-related gene sets, and significantly associated with high infiltration fraction of stromal cells, including fibroblasts, adipocytes (all p&lt;0.001). On the other hand, they significantly enriched multiple pro-cancerous gene sets, such as TGF-β signaling, hypoxia, and angiogenesis, consistent with infiltration of angiogenesis-related stromal cells such as microvascular and lymphatic endothelial cells, and pericytes (all p&lt;0.001). These results were shown consistently in two large cohort; TCGA and GSE84437. Conclusions: EMT signature in a bulk tumor was associated with cancer aggravating pathways such as TGF-β signaling, hypoxia, and angiogenesis, as well as with worse survival in gastric cancer patients.

Spatially resolved multi-region transcriptomic subtyping and assessment of gene expression profiles associated with long-term benefit from chemo-immunotherapy in patients with extensive-stage small cell lung cancer (ES-SCLC).

8594 Background: Transcriptomic subtyping holds promise for personalized therapy in SCLC, but intratumoral transcriptomic heterogeneity and its clinical significance remain poorly defined. In this study, we aimed to assess transcription factor defined subtypes within multiple regions of intact tissues and identify gen sets associated with long-term chemo-immunotherapy benefit. Methods: We assessed baseline FFPE tumors from 32 ES-SCLC patients enrolled in the IMfirst clinical trial (EudraCT: 2019-002784-10). We used GeoMx DSP to perform transcriptomic analysis from multiple intratumoral regions of interest (ROIs). We used an assay with +1800 genes (GeoMx CTA) plus custom-designed mRNA probes targeting subtype-defining transcription factors not included in the CTA assay (POU2F3, NEUROD1, and YAP1). Custom probes were quantitatively validated using FFPE cell lines. Results: We profiled 175 ROIs from 32 tumors. Cluster analysis based on the expression of ASCL1, NEUROD1, POU2F3, and YAP1 showed that all samples clustered within 4 groups: SCLC-A (76 ROIs [43%]), SCLC-N (31 ROIs [18%]), SCLC-P (18 ROIs [10%]), and SCLC-Y (50 ROIs [29%]). ASCL1 was the most abundantly expressed transcription factor, prevailed in the SCLC-A subtype, and showed inverse correlation with POU2F3 (r=-0.55, p&lt;0.0001) and YAP1(r=-0.70, p&lt;0.0001). YAP1 was expressed at low levels and was more broadly distributed across all 4 subtypes. Differential expression and gene set enrichment analysis (GSEA) using linear mixed models revealed that SCLC-A subtype was enriched in cell cycle and DNA damage repair genes, and showed repression of multiple gene sets associated with anti-tumor immune response. In contrast, SCLC-Y subtype showed the opposite pattern. The SCLC-P subtype was also enriched in genes related to cancer antigens and T-cell checkpoints. Most patients (n=18, 56%) had tumors with coexistence of more than one subtype, not evident through morphological inspection. Combined SCLC-A and SCLC-Y subtypes was the most common mixed phenotype (n=8, 25%). Four patients (13%) had tumors with co-existence of three subtypes. Transcriptional subtypes, subtype-defining transcription factors as single genes, or the presence of subtype heterogeneity, were not associated with outcomes. Gene sets involved in mitochondrial metabolism and MHC class I antigen presentation were the highest enriched pathways in tumors from patients with sustained benefit (time to progression ³ 12 months). Conclusions: This study reveals substantial intratumoral transcriptomic subtype heterogeneity in human SCLC. In this limited sample set, we did not observe outcome association for transcriptional subtypes. Our findings related to long-term chemo-immunotherapy benefit require validation in independent cohorts.

MRPL51 is a downstream target of FOXM1 in promoting the malignant behaviors of lung adenocarcinoma.

Mitochondrial ribosome protein L51 (MRPL51) is a 39S subunit protein of the mitochondrial ribosome. Its dysregulation may be involved in non-small cell lung cancer. The present study aimed to explore MRPL51 expression in lung adenocarcinoma (LUAD) and normal lung tissues, as well as its regulatory effects on malignant LUAD behaviors. In addition, the role of forkhead box protein M1 (FOXM1) in MRPL51 transcription was studied. Bioinformatics analysis and subsequent in vitro experiments, including western blotting, immunofluorescent staining, Transwell invasion assay, dual-luciferase assay and chromatin immunoprecipitation quantitative PCR were conducted. The results demonstrated that MRPL51 expression was upregulated at both the mRNA and protein levels in LUAD tissues compared with normal lung tissues. Gene Set Enrichment Analysis demonstrated that LUAD tissues with higher MRPL51 expression also had higher expression levels of genes enriched in multiple gene sets, including 'DNA_REPAIR', 'UNFOLDED_PROTEIN_RESPONSE', 'MYC_TARGETS_V1', 'OXIDATIVE_ PHOSPHORYLATION', 'MTORC1_SIGNALING', 'REACTIVE_OXYGEN_SPECIES_PATHWAY', 'MYC_ TARGETS_V2', 'E2F_TARGETS' and 'G2M_ CHECKPOINT'. MRPL51 expression was positively correlated with 'cell cycle', 'DNA damage', 'DNA repair', epithelial-mesenchymal transition ('EMT'), 'invasion' and 'proliferation' of LUAD cells at the single-cell level. Compared to the negative control, MRPL51 knockdown decreased N-cadherin and vimentin expression but increased E-cadherin expression in A549 and Calu-3 cells. MRPL51 knockdown suppressed cell proliferation, induced G1 phase arrest and decreased cell invasion. Patients with LUAD and higher MRPL51 expression had a significantly shorter overall survival (OS). FOXM1 could bind to the MRPL51 gene promoter and activate its transcription. In conclusion, MRPL51 was transcriptionally activated by FOXM1 in LUAD and contributed to the malignant behaviors of tumor cells, including EMT, cell cycle progression and invasion. High MRPL51 expression may be a prognostic biomarker indicating poor OS.

Identification of Potential Drug Targets in Erythrocyte Invasion Pathway of Plasmodium falciparum.

The erythrocyte invasion phase plays a critical role in multiplication, sexual determination, and drug resistance in Plasmodium falciparum. In order to identify the critical genes and pathways in the erythrocyte invasion phase, the gene set (GSE129949) and the RNA-Seq count data for the W2mef strain were used for further analysis. An integrative bioinformatics study was performed to scrutinize genes as potential drug targets. 487 differentially expressed genes (DEGs) with an adjusted P value < 0.001 enriched 47 Gene Ontology (GO) terms that were over-represented based on hyper-geometric analysis P value < 0.01. Protein-Protein interaction network analysis was done using DEGs with higher confidence interactions (PPI score threshold = 0.7). MCODE and cytoHubba apps were utilized to define the hub proteins and rank them based on multiple topological analyses and MCODE scores. Furthermore, Gene Set Enrichment Analysis (GSEA) was carried out by using 322 gene sets from the MPMP database. The genes involved in multiple significant gene sets were determined by leading-edge analysis. Our study identified six genes encoding proteins that could be potential drug targets involved in the erythrocyte invasion phase related to merozoites motility, cell-cycle regulation, G-dependent protein kinase phosphorylation in schizonts, control of microtubule assembly, and sexual commitment. The druggability of those proteins was calculated based on the DCI (Drug Confidence Index) and predicted binding pockets' values. The protein that showed the best binding pocket value was subjected to deep learning-based virtual screening. The study identified the best small molecule inhibitors in terms of drug-binding score against the proteins for inhibitor identification.