Differential Pathway Activation Research Articles

Abstract LB435: Large panel organoid drug testing combined with biomarker analysis: Unveiling prospects for mechanism of action identification and preclinical patient stratification

Abstract Introduction The development of oncology drugs has a persistently high attrition rate of greater than 95%, requiring the development and implementation of novel models and technologies aimed at better predictive abilities during the preclinical phase. Among these, organoid technologies, which involve propagating tumor stem cells in a near-native state, have proven to markedly advance drug testing with enhanced precision. Our previously developed assay-ready organoid technology has paved the way for large panel organoid screening, allowing organoids to augment the efficiency of drug development pipelines, boost patient-derived model usage in preclinical studies, and facilitate accurate predictions of drug action mechanisms. Here, we present a proof-of-concept (POC) large scale organoid experiment, studying the responses of 50 models to the cytotoxic agent paclitaxel, using next generation sequencing data to analyze the drug mechanism of action (MOA) and identify biomarkers that predict drug responses. Methods A panel of 50 organoid models was characterized for paclitaxel response in 9-dose-response curves by measuring ATP levels after a 5-day exposure. Models were genomically profiled by whole-exome sequencing (n = 47) and whole-transcriptome sequencing (n = 44) to analyze the association between organoid pharmacology data and genomic information and elucidate genes and pathways that may be related with, or predictive of, the treatment responses. We performed biomarker analysis on gene expression, mutation, and copy number alteration levels. Results By assessing organoid sensitivity to paclitaxel, we observed no significant indication-specific difference in organoid responses across 7 cancer types. The 50 models were then categorized into responders (AUC < 1.5; n=15) and non-responders (AUC > 2.6; n=14) for biomarker discovery. Differential expression analysis and pathway activation analysis revealed genes and pathways involved in the cell cycle and DNA replication. DNA recombination differentiates responders from non-responders, while genes involved in epithelial cell proliferation, differentiation, and wound healing are associated with drug resistance. Mutation of proto-oncogene RB Transcriptional Corepressor 1, RB1 (a negative regulator of the cell cycle), was found to be a biomarker enriched in non-responders to paclitaxel, corroborating earlier clinical findings. Using the identified profile, we predicted responses for non-tested models in our biobank and validated our approach. Conclusion Large panel organoid drug screening in combination with biomarker analysis brings great opportunities for increasing drug pipeline efficiency. The POC here presented highlights how such a screen can identify MOAs and identify relevant biomarker profiles to be used in clinical trial patient stratification. Citation Format: Linda Xue, Liza Wijler, Marten Hornsveld, Sheng Guo, Leo Price, Marrit Putker, Ludovic Bourre. Large panel organoid drug testing combined with biomarker analysis: Unveiling prospects for mechanism of action identification and preclinical patient stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB435.

Abstract 1743: Pan-cancer review of TP53 somatic alterations as a function of tumor type: Sometimes lost, sometimes gained, frequently altered

Abstract Background: TP53 is the gene most frequently disrupted by somatic alterations in cancer. Mutations include single nucleotide variants resulting in functional alterations, as well as premature stop codons, splice variants, and cryptic alterations. TP53 gene dosage is disrupted by heterozygous and homozygous deletions, copy-neutral loss of heterozygosity, and other structural changes such as intragenic deletions and fusions. TP53 is altered by epigenetic events and viral oncogenes, such as E6 expressed by the human papillomavirus. The readout of altered TP53 includes decreased gene expression of either variant or wild-type alleles. Epistatic alteration of TP53 pathway components have also been described, with the E3 ubiquitin ligase MDM2 being the canonical example. Depending on the type of TP53 alteration, there may be differential signaling consequences, ranging from loss of function and dominant negative to oncogenic signaling. Hypothesis: Patterns of TP53 mutations will vary based on tumor type. Differential alterations of TP53 will suggest differential pathway dependence and downstream pathophysiology that may be relevant to signaling and therapeutic potential. Methods: We analyzed extensive cancer genomics repositories, such as the Pan-Cancer Atlas (10,000+ tumors, >30 cancer types) from The Cancer Genome Atlas project and the International Cancer Genome Consortium. Assessing genes in the TP53 pathway, we examined somatic mutations, copy number alterations, structural changes, and gene expression. Epigenetic data was considered when available. Results: A significant pattern amongst sarcomas, prostate cancers, and urothelial carcinomas emerged, revealing that certain tumors exhibited a predominant classic tumor suppressor gene genotype. This genotype featured frequent homozygous deletions, inactivating mutations, and relatively low TP53 gene expression, often accompanied by epistatic MDM2 amplification. By contrast, other tumors demonstrate an atypical pattern in which TP53 mutations were rarely if ever associated with loss of TP53 gene expression with low levels of homozygous TP53 gene deletion. Likewise, atypical TP53 mutant tumor types rarely demonstrated MDM2 amplifications, and in opposition to typical TP53 cases, MDM2 amplification was positively associated with TP53 mutation rather than anti-correlated. Head and neck squamous cell carcinoma, uterine carcinoma, and pancreatic adenocarcinoma followed this pattern. Investigation within selected tumor types such as lung squamous cell carcinoma suggested that both typical and atypical TP53 mutation patterns could be observed. Conclusions: TP53 mutation patterns were strongly associated with tumor type and may suggest differential pathway activation according to the somatic alteration status. Citation Format: Ubaid A. Tanveer, David N. Hayes, Julie George, Jeremiah R. Holt, Martin Peifer, Roman Thomas. Pan-cancer review of TP53 somatic alterations as a function of tumor type: Sometimes lost, sometimes gained, frequently altered [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 1743.

Elucidating T cell dynamics and molecular mechanisms in syngeneic and allogeneic islet transplantation through single-cell RNA sequencing.

Islet transplantation is a promising therapy for diabetes treatment. However, the molecular underpinnings governing the immune response, particularly T-cell dynamics in syngeneic and allogeneic transplant settings, remain poorly understood. Understanding these T cell dynamics is crucial for enhancing graft acceptance and managing diabetes treatment more effectively. This study aimed to elucidate the molecular mechanisms, gene expression differences, biological pathway alterations, and intercellular communication patterns among T-cell subpopulations after syngeneic and allogeneic islet transplantation. Using single-cell RNA sequencing, we analyzed cellular heterogeneity and gene expression profiles using the Seurat package for quality control and dimensionality reduction through t-SNE. Differentially expressed genes (DEGs) were analyzed among different T cell subtypes. GSEA was conducted utilizing the HALLMARK gene sets from MSigDB, while CellChat was used to infer and visualize cell-cell communication networks. Our findings revealed genetic variations within T-cell subpopulations between syngeneic and allogeneic islet transplants. We identified significant DEGs across these conditions, highlighting molecular discrepancies that may underpin rejection or other immune responses. GSEA indicated activation of the interferon-alpha response in memory T cells and suppression in CD4+ helper and γδ T cells, whereas TNFα signaling via NFκB was particularly active in regulatory T cells, γδ T cells, proliferating T cells, and activated CD8+ T cells. CellChat analysis revealed complex communication patterns within T-cell subsets, notably between proliferating T cells and activated CD8+ T cells. In conclusion, our study provides a comprehensive molecular landscape of T-cell diversity in islet transplantation. The insights into specific gene upregulation in xenotransplants suggest potential targets for improving graft tolerance. The differential pathway activation across T-cell subsets underscores their distinct roles in immune responses posttransplantation.

Human Blood Serum Can Diminish EGFR-Targeted Inhibition of Squamous Carcinoma Cell Growth through Reactivation of MAPK and EGFR Pathways.

Regardless of the presence or absence of specific diagnostic mutations, many cancer patients fail to respond to EGFR-targeted therapeutics, and a personalized approach is needed to identify putative (non)responders. We found previously that human peripheral blood and EGF can modulate the activities of EGFR-specific drugs on inhibiting clonogenity in model EGFR-positive A431 squamous carcinoma cells. Here, we report that human serum can dramatically abolish the cell growth rate inhibition by EGFR-specific drugs cetuximab and erlotinib. We show that this phenomenon is linked with derepression of drug-induced G1S cell cycle transition arrest. Furthermore, A431 cell growth inhibition by cetuximab, erlotinib, and EGF correlates with a decreased activity of ERK1/2 proteins. In turn, the EGF- and human serum-mediated rescue of drug-treated A431 cells restores ERK1/2 activity in functional tests. RNA sequencing revealed 1271 and 1566 differentially expressed genes (DEGs) in the presence of cetuximab and erlotinib, respectively. Erlotinib- and cetuximab-specific DEGs significantly overlapped. Interestingly, the expression of 100% and 75% of these DEGs restores to the no-drug level when EGF or a mixed human serum sample, respectively, is added along with cetuximab. In the case of erlotinib, EGF and human serum restore the expression of 39% and 83% of DEGs, respectively. We further assessed differential molecular pathway activation levels and propose that EGF/human serum-mediated A431 resistance to EGFR drugs can be largely explained by reactivation of the MAPK signaling cascade.

Plasma Proteomic Patterns Show Sex Differences in Early Concentric Left Ventricular Remodeling.

Concentric remodeling (cRM) can precede heart failure with preserved ejection fraction (HFpEF), a condition prevalent in women. Patients (n=60 593, 54.2% women) visiting outpatient clinics of Cardiology Centers of the Netherlands were analyzed for cRM, HFpEF development, and mortality risk. We studied risk factors for relative wall thickness both sex-stratified and in women and men combined. Biomarker profiling was performed (4534 plasma proteins) in a substudy involving 557 patients (65.4% women) to identify pathways involved in cRM. cRM was present in 23.5% of women and 27.6% of men and associated with developing HFpEF (HR, 2.15 [95% CI, 1.51-2.99]) and mortality risk (HR, 1.09 [95% CI, 1.00-1.19]) in both sexes. Age, heart rate, and hypertension were statistically significantly stronger risk factors for relative wall thickness in women than men. Higher circulating levels of IFNA5 (interferon alpha-5) were associated with higher relative wall thickness in women only. Pathway analysis revealed differential pathway activation by sex and increased expression of inflammatory pathways in women. cRM is prevalent in approximately 1 in 4 women and men visiting outpatient cardiology clinics and associated with HFpEF development and mortality risk in both sexes. Known risk factors for cRM were more strongly associated in women than men. Proteomic analysis revealed inflammatory pathway activation in women, with a central role for IFNA5. Differential biologic pathway activation by sex in cRM may contribute to the female predominance of HFpEF and holds promise for identification of new therapeutic avenues for prevention and treatment of HFpEF. URL: https://www. gov; Unique identifier: NCT001747.

Transcriptome profile changes in the jejunum of nonhuman primates exposed to supralethal dose of total- or partial-body radiation

The risk of exposure of the general public or military personnel to high levels of ionizing radiation from nuclear weapons or radiological accidents is a dire national security matter. The development of advanced molecular biodosimetry methods, those that measure biological response, such as transcriptomics, to screen large populations of radiation-exposed victims is key to improving survival outcomes during radiological mass casualty scenarios. In this study, nonhuman primates were exposed to either 12.0 Gy cobalt-60 gamma (total-body irradiation, TBI) or X-ray (partial-body irradiation, PBI) 24 h after administration of a potential radiation medical countermeasure, gamma-tocotrienol (GT3). Changes in the jejunal transcriptomic profiles in GT3-treated and irradiated animals were compared to healthy controls to assess the extent of radiation damage. No major effect of GT3 on radiation-induced transcriptome at this radiation dose was identified. About 80% of the pathways with a known activation or repression state were commonly observed between both exposures. Several common pathways activated due to irradiation include FAK signaling, CREB signaling in the neurons, phagosome formation, and G-protein coupled signaling pathway. Sex-specific differences associated with excessive mortality among irradiated females were identified in this study, including Estrogen receptor signaling. Differential pathway activation was also identified across PBI and TBI, pointing towards altered molecular response for different degrees of bone marrow sparing and radiation doses. This study provides insight into radiation-induced changes in jejunal transcriptional profiles, supporting the investigation for the identification of biomarkers for radiation injury and countermeasure efficacy.

P020 Mapping the changing intercellular communication and its downstream effect in Ulcerative Colitis

Abstract Background Intercellular communication is essential for growing and differentiating in multicellular organisms by transducing the signal from cell to cell. Despite its importance, the molecular background is less discovered due to the lack of data. This gap has started to be addressed with the appearance of single-cell omics approaches providing an insight among others into the gene expression of individual cells. Methods We have developed a method to predict and compare cell-cell signalling interactions using single-cell RNAseq data from colon biopsies. Transcriptomic data alone is not capable of connecting the cells, a reliable network resource is needed to mediate the signal via protein-protein interactions between the source and target cells. Here we used OmniPath - a resource providing not only intra- and intercellular interactions but also annotations of proteins involved in the interplay of cells - to reconstruct signalling networks. We examined intercellular communication among five cell-types (regulatory T cell, macrophage, dendritic cell, goblet cell and myofibroblast) in healthy colon and during Ulcerative Colitis. Results Our analysis shows that there are significant differences in the type of cell-cell communication (ligand-receptor connections, adherens junctions, etc.) between the healthy and Ulcerative Colitis (UC) conditions, and these differences lead to altered downstream effects in the signal receiving cell. In both conditions, the ligand-receptor and adhesion connections were overrepresented, however cell junctions were less abundant in UC. Regarding the communication among the five cell-types, in healthy condition, cells are tightly connected to dendritic cells while in diseased condition to regulatory T cells. Focusing on ligand-receptor interactions between myofibroblasts and regulatory T cells, our pipeline identified the MAPK, Toll-like receptor (TLR) 2/6 and TLR 7/8 pathways enriched downstream in healthy conditions. In contrast, TLR3 and TLR4 pathways were affected by the myofibroblast in Ulcerative Colitis. Conclusion We found key intercellular mechanisms leading to well-defined differential pathway activation profiles. We showed that in uninflamed UC condition myofibroblasts disrupt the anti-inflammatory effect of regulatory T cells. Our pipeline is able to predict and analyse cell-cell interactions and their downstream effects and to highlight the differences in healthy and diseased states.

Hedgehog gene expression patterns among intrinsic subtypes of breast cancer: Prognostic relevance

PurposeHedgehog (Hh) signaling is a crucial developmental regulatory pathway recognized as a primary oncogenesis driver in various human cancers. However, its role in breast carcinoma (BC) has been underexplored. MethodsWe analyzed the expression of several Hh associated genes in a clinical series and breast cancer cell lines. We included 193 BC stratified according to intrinsic immunophenotypes. Gene expression profiling ofBOC, PTCH, SMO, GLI1, GLI2, and GLI3 was performed by qRT-PCR. Results were correlated with clinical-pathological variables and outcome. ResultsWe observed expression ofGLI2 in triple-negative/basal-like (TN/BL) and GLI3 in luminal cells. In samples, BOC, GLI1, GLI2, and GLI3 expression correlated significantly with luminal tumors and good prognostic factors. In contrast, PTCH and SMO correlated with TN/BL phenotype and nodal involvement. Patients whose tumors expressed SMO had a poorer outcome, especially those with HER2 phenotype. Positive lymph-node status and high SMO remained independent poor prognostic factors. ConclusionOur results support a differential Hh pathway activation in BC phenotypes.SMO levels stratified patients at risk of recurrence and death in HER2 phenotype, and it showed an independent prognostic value. Therefore, SMO could be a potential therapeutic target for a subset of BC patients.

Distinct Single Cell Gene Expression in Peripheral Blood Monocytes Correlates With Tumor Necrosis Factor Inhibitor Treatment Response Groups Defined by Type I Interferon in Rheumatoid Arthritis.

Previously, we demonstrated in test and validation cohorts that type I IFN (T1IFN) activity can predict non-response to tumor necrosis factor inhibitors (TNFi) in rheumatoid arthritis (RA). In this study, we examine the biology of non-classical and classical monocytes from RA patients defined by their pre-biologic treatment T1IFN activity. We compared single cell gene expression in purified classical (CL, n = 342) and non-classical (NC, n = 359) monocytes. In our previous work, RA patients who had either high IFNβ/α activity (>1.3) or undetectable T1IFN were likely to have EULAR non-response to TNFi. In this study comparisons were made among patients grouped according to their pre-biologic treatment T1IFN activity as clinically relevant: “T1IFN undetectable (T1IFN ND) or IFNβ/α >1.3” (n = 9) and “T1IFN detectable but IFNβ/α ≤ 1.3” (n = 6). In addition, comparisons were made among patients grouped according to their T1IFN activity itself: “T1IFN ND,” “T1IFN detected and IFNβ/α ≤ 1.3,” and “IFNβ/α >1.3.” Major differences in gene expression were apparent in principal component and unsupervised cluster analyses. CL monocytes from the T1IFN ND or IFNβ/α >1.3 group were unlikely to express JAK1 and IFI27 (p < 0.0001 and p 0.0005, respectively). In NC monocytes from the same group, expression of IFNAR1, IRF1, TNFA, TLR4 (p ≤ 0.0001 for each) and others was enriched. Interestingly, JAK1 expression was absent in CL and NC monocytes from nine patients. This pattern most strongly associated with the IFNβ/α>1.3 group. Differences in gene expression in monocytes among the groups suggest differential IFN pathway activation in RA patients who are either likely to respond or to have no response to TNFi. Additional transcripts enriched in NC cells of those in the T1IFN ND and IFNβ/α >1.3 groups included MYD88, CD86, IRF1, and IL8. This work could suggest key pathways active in biologically defined groups of patients, and potential therapeutic strategies for those patients unlikely to respond to TNFi.

Transcriptional Regulation of INSR, the Insulin Receptor Gene.

The insulin receptor gene encodes an evolutionarily conserved signaling protein with a wide spectrum of functions in metazoan development. The insulin signaling pathway plays key roles in processes such as metabolic regulation, growth control, and neuronal function. Misregulation of the pathway features in diabetes, cancer, and neurodegenerative diseases, making it an important target for clinical interventions. While much attention has been focused on differential pathway activation through ligand availability, sensitization of overall signaling may also be mediated by differential expression of the insulin receptor itself. Although first characterized as a “housekeeping” gene with stable expression, comparative studies have shown that expression levels of the human INSR mRNA differ by tissue and in response to environmental signals. Our recent analysis of the transcriptional controls affecting expression of the Drosophila insulin receptor gene indicates that a remarkable amount of DNA is dedicated to encoding sophisticated feedback and feed forward signals. The human INSR gene is likely to contain a similar level of transcriptional complexity; here, we summarize over three decades of molecular biology and genetic research that points to a still incompletely understood regulatory control system. Further elucidation of transcriptional controls of INSR will provide the basis for understanding human genetic variation that underlies population-level physiological differences and disease.

Oncogenic Properties of the Antisense lncRNA COMET in BRAF- and RET-Driven Papillary Thyroid Carcinomas.

RET rearrangements as well as BRAF and RAS mutations drive differential pathway activation in papillary thyroid carcinomas, leading to different tumor phenotypes and prognoses. Although The Cancer Genome Atlas Consortium has identified tumor subgroups based on protein-coding gene signatures, neither expression of long noncoding RNAs (lncRNA) nor their correlation with specific tumor-driving mutations and rearrangements have been systematically assessed. Here, we reanalyzed our RNA-sequencing data using a de novo discovery approach to identify lncRNAs and define tumor subtype-specific signatures of annotated lncRNAs. Among them, we identified COMET (Correlated-to-MET), a natural antisense transcript that was highly expressed in carcinomas harboring BRAF V600E mutation or RET gene rearrangements (i.e., BRAF-like tumors) and induced the downstream MAPK pathway. In papillary thyroid carcinomas, COMET was part of a coexpression network including different oncogenes belonging to the MAPK pathway, and its expression highly correlated with MET expression. Depletion of COMET resulted in reduced expression of genes within this network, including the MET oncogene. COMET repression inhibited viability and proliferation of tumor cells harboring BRAF V600E somatic mutation or RET oncogene rearrangement and dramatically reduced motility and invasiveness of tumor cells. Moreover, silencing COMET markedly increased sensitivity to vemurafenib, a common inhibitor of mutated B-raf. Collectively, our results suggest COMET as a new target to improve drug-based cancer therapies, especially in BRAF-mutated and MET-addicted papillary thyroid carcinomas. SIGNIFICANCE: These results highlight the oncogenic role of lncRNA COMET in thyroid and indicate it as a potential new target to overcome vemurafenib resistance in BRAF-mutated and MET-addicted carcinomas.

3521 Distinct single cell gene expression in peripheral blood monocytes correlates with treatment response groups to TNF-alpha inhibition in rheumatoid arthritis

OBJECTIVES/SPECIFIC AIMS: The cellular mechanisms that underlie the IFNβ/α ratio that predicts response are not known. Effects of IFN on single immune cells may be masked in whole blood or mixed cell populations. By studying the effect of IFNβ/α activity ratio on individual monocytes, we can determine the functional impact of the IFN ratio and suggest the cellular mechanisms that underlie response/non-response to TNFi therapy in RA. METHODS/STUDY POPULATION: We used single cell analysis to investigate whether monocyte gene expression differs significantly between RA patients according to their pre-TNFi serum IFN-β/α ratio. Single classical (CL) and non-classical (NC) blood-derived monocytes were isolated from 15 seropositive RA subjects prior to biologic therapy. Subjects were grouped by pre-TNFi serum IFN-β/α ratio into two groups, those with a high IFN-β/α ratio (≥1.3, n = 6) and those with a low IFN-β/α ratio (&lt;1.3, n = 9). 87 target genes were analyzed. Genes that varied significantly between the groups by categorical analyses were tested in multivariate logistic regression models. RESULTS/ANTICIPATED RESULTS: Every participant was seropositive for rheumatoid factor and antibodies to cyclic citrullinated peptide. Among the participants in the groups, there were no significant differences in age or DAS scores (P&gt;0.05). The treatments were comparable and none were being treated with biologic therapy. There were striking differences in monocyte gene expression between patients with pre-treatment blood IFNβ/α activity &lt;1.3 and ≥1.3. Expression of (1) key type I IFN pathway genes (JAK1, STAT2, IFIT2, IFIH1, PRDM1); (2) IL12; (3) CD36; and (4) CTLA4 were the strongest differentiators between groups (p&lt;0.0001 for each, corrected for multiple comparisons). DISCUSSION/SIGNIFICANCE OF IMPACT: In this study we were able to measure gene expression in single monocytes from seropositive RA patients prior to biologic treatment. Within-cell co-expression patterns demonstrate biological differences in monocytes of RA patients with an IFNβ/α ≥1.3, the ratio of type I IFNs which predicts non-response to TNFi. The data suggest that there may be differential IFN production and pathway activation in patients who do not respond to TNFi. The increased expression of CD36 in monocytes from RA patients with high IFN β/α activity may be a reflection of increased “foam cells” in the inflamed tissue of patients who do not respond to TNFi. Enrichment of CTLA4 in those with high serum IFNβ/α suggests that CTLA4-Ig may be less likely to be an effective alternative for someone who is not likely to respond to TNFi. Current work includes determining whether the peripheral blood findings reflect altered cellular composition, type I IFN production and signaling in the synovium. Significance: This work will help to develop a more individualized approach to therapy in RA and determine an immunological basis of response/non-response to TNFi.

Indirect monitoring of TORC1 signalling pathway reveals molecular diversity among different yeast strains.

Saccharomyces cerevisiae is the main species responsible for the alcoholic fermentation in wine production. One of the main problems in this process is the deficiency of nitrogen sources in the grape must, which can lead to stuck or sluggish fermentations. Currently, yeast nitrogen consumption and metabolism are under active inquiry, with emphasis on the study of the TORC1 signalling pathway, given its central role responding to nitrogen availability and influencing growth and cell metabolism. However, the mechanism by which different nitrogen sources activates TORC1 is not completely understood. Existing methods to evaluate TORC1 activation by nitrogen sources are time-consuming, making difficult the analyses of large numbers of strains. In this work, a new indirect method for monitoring TORC1 pathway was developed on the basis of the luciferase reporter gene controlled by the promoter region of RPL26A gene, a gene known to be expressed upon TORC1 activation. The method was tested in strains representative of the clean lineages described so far in S.cerevisiae. The activation of the TORC1 pathway by a proline-to-glutamine upshift was indirectly evaluated using our system and the traditional direct methods based on immunoblot (Sch9 and Rps6 phosphorylation). Regardless of the different molecular readouts obtained with both methodologies, the general results showed a wide phenotypic variation between the representative strains analysed. Altogether, this easy-to-use assay opens the possibility to study the molecular basis for the differential TORC1 pathway activation, allowing to interrogate a larger number of strains in the context of nitrogen metabolism phenotypic differences.

Sex-specific differences in primary neonatal murine lung fibroblasts exposed to hyperoxia in vitro: implications for bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of the neonate characterized by impaired alveolarization and vascular growth. BPD is more common in premature male infants, but the reasons underlying sexually dimorphic outcomes are not known. It is thought that alterations in fibroblast phenotype in response to environmental stress such as hyperoxia contribute to BPD. Notch signaling creates a profibrotic environment in the lung. However, the role of hyperoxia on differential Notch pathway activation in male vs. female neonatal lung fibroblasts is not known. Primary murine lung fibroblasts from 10-day-old male and female mice were exposed to room air (21% O2, 5% CO2) or hyperoxia (95% O2, 5% CO2), and changes in cell proliferation, viability and expression of fibrosis-related genes and Notch pathway mediators were measured. Upon exposure to hyperoxia, cell proliferation was arrested in male and female fibroblasts, but cell viability was preserved. Increased Notch pathway activation was noted in male fibroblasts along with differential sex-specific modulation of key Notch pathway mediators in response to hyperoxia. α-Smooth muscle actin expression was increased in both male and female fibroblasts upon exposure to hyperoxia. Male and female fibroblasts further demonstrated distinct changes in expression of key fibrosis-related genes upon exposure to hyperoxia. Differential Notch pathway activation and distinct differences in the expression of key fibrosis-related genes might contribute to the sex-specific differences seen in hyperoxia-induced fibrosis and inhibition of lung development in BPD, with more severe implications in male neonates.

Multiparametric Magnetic Resonance Imaging Features Identify Aggressive Prostate Cancer at the Phenotypic and Transcriptomic Level

Multiparametric magnetic resonance imaging is a diagnostic tool for prostate cancer with limited data on prognostic use. We sought to determine whether multiparametric magnetic resonance could predict aggressive prostate cancer features. We retrospectively analyzed the records of 206 patients who underwent radical prostatectomy between 2013 and 2017. All patients had available RNA expression data on the final pathology specimen obtained from a location corresponding to a lesion location on multiparametric magnetic resonance imaging. The association between the PIRADS™ (Prostate Imaging Reporting and Data System) score and adverse pathology features were analyzed. We also performed differential transcriptomic analysis between the PIRADS groups. Factors associated with adverse pathology were analyzed using a multivariable logistic regression model. Lesion size (p = 0.03), PIRADS score (p = 0.02) and extraprostatic extension (p = 0.01) associated significantly with the Decipher® score. Multivariable analysis showed that the PIRADS score (referent PIRADS 3, OR 8.1, 95% CI 1.2-57.5, p = 0.04), the Gleason Grade Group (referent 3, OR 5.6, 95% CI 1.5-21.1, p = 0.01) and prostate specific antigen (OR 1.103, 95% CI 1.011-1.203) were risk factors for adverse pathology findings. The difference between PIRADS 4 and 5 did not reach significance (OR 1.9, 95% CI 0.8-4.5, p = 0.12). However, the PI3K-AKT-mTOR, WNT-β and E2F signaling pathways were more active in PIRADS 5 than in PIRADS 4 cases. The PIRADS score is associated with adverse pathology results, increased metastatic risk and differential genomic pathway activation.

Therapeutic opportunities for alcoholic steatohepatitis and nonalcoholic steatohepatitis: exploiting similarities and differences in pathogenesis.

Alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH) are among the most frequent causes of chronic liver disease in the United States. Although the two entities are triggered by different etiologies - chronic alcohol consumption (ASH) and obesity-associated lipotoxicity (NASH) - they share overlapping histological and clinical features owing to common pathogenic mechanisms. These pathogenic processes include altered hepatocyte lipid metabolism, organelle dysfunction (i.e., ER stress), hepatocyte apoptosis, innate immune system activation, and hepatic stellate cell activation. Nonetheless, there are several disease-specific molecular signaling pathways, such as differential pathway activation downstream of TLR4 (MyD88-dependence in NASH versus MyD88-independence in ASH), inflammasome activation and IL-1β signaling in ASH, insulin resistance and lipotoxicity in NASH, and dysregulation of different microRNAs, which clearly highlight that ASH and NASH are two distinct biological entities. Both pathogenic similarities and differences have therapeutic implications. In this Review, we discuss these pathogenic mechanisms and their therapeutic implications for each disease, focusing on both shared and distinct targets.

Abstract 457: Rad-deletion Provides Dual Therapeutic Benefits for the Heart

Rationale: Myocardial infarction (MI) compromises the overall mechanical function of the heart, reducing cardiac output and triggering decompensatory remodeling. Rad GTPase is an L-type calcium channel complex (LTCC) constituent that governs trigger calcium in the myocardium. Rad deletion in mice results in increased Ca 2+ handling and a sustained non-pathological improvement in left ventricular function compared to wildtype. Hypothesis: Rad-ablation attenuates post-ischemic loss of function, resulting in reduced remodeling and improved long-term contractility. Methods and Results: We subjected Rad-deficient (Rad -/- ) mice to ligation of the left anterior descending (LAD) coronary artery, and monitored cardiac structure and function using echocardiography and single cell Ca 2+ measurements. We found that Rad deletion reduces both mortality and contractile dysfunction after MI, as well as ventricular dilation over five weeks. This improvement is also accompanied by preserved Ca 2+ handling in isolated myocytes. Histological and MRI examination of ex vivo global ischemia and in vivo 24 hour post-MI myocardium revealed that initial infarct size and area at risk are comparable between knockout and wildtype. Rad loss reduced scar spread independent of preserving tissue viability. mRNA microarray findings implicated differential inflammatory pathway activation with Rad-deletion. Rad -/- showed reduced neutrophil extravasation and reduced inflammatory cytokines in the left ventricle 24 hours after MI. Conclusion: Rad deletion results in reduced cardiac remodeling, diminished myocardial inflammation, and improved contractile function before and after MI. These results suggest that Rad-deletion is a novel therapeutic direction that may serve as a combined positive inotrope and regulator of acute MI inflammatory response.

Disruption of KIF3A in patient-derived glioblastoma cells: effects on ciliogenesis, hedgehog sensitivity, and tumorigenesis.

KIF3A, a component of the kinesin-2 motor, is necessary for the progression of diverse tumor types. This is partly due to its role in regulating ciliogenesis and cell responsiveness to sonic hedgehog (SHH). Notably, primary cilia have been detected in human glioblastoma multiforme (GBM) tumor biopsies and derived cell lines. Here, we asked whether disrupting KIF3A in GBM cells affected ciliogenesis, in vitro growth and responsiveness to SHH, or tumorigenic behavior in vivo. We used a lentiviral vector to create three patient-derived GBM cell lines expressing a dominant negative, motorless form of Kif3a (dnKif3a). In all unmodified lines, we found that most GBM cells were capable of producing ciliated progeny and that dnKif3a expression in these cells ablated ciliogenesis. Interestingly, unmodified and dnKif3a-expressing cell lines displayed differential sensitivities and pathway activation to SHH and variable tumor-associated survival following mouse xenografts. In one cell line, SHH-induced cell proliferation was prevented in vitro by either expressing dnKif3a or inhibiting SMO signaling using cyclopamine, and the survival times of mice implanted with dnKif3a-expressing cells were increased. In a second line, expression of dnKif3a increased the cells' baseline proliferation while, surprisingly, sensitizing them to SHH-induced cell death. The survival times of mice implanted with these dnKif3a-expressing cells were decreased. Finally, expression of dnKif3a in a third cell line had no effect on cell proliferation, SHH sensitivity, or mouse survival times. These findings indicate that KIF3A is essential for GBM cell ciliogenesis, but its role in modulating GBM cell behavior is highly variable.

ID: 122: The gp130 cytokines mediate differential signal pathway activation and transcriptional responses in astrocytes versus microglia

The gp130 cytokines include IL-6, IL-11, LIF and OSM. These cytokines have important roles in neuroinflammation and neurodevelopment. However, the nature of any cell-specific responses of astrocytes and microglia to this cytokine family and their molecular basis is not well understood. Here, the gp130 family cytokine signal pathway activation and global transcriptional responses were examined in murine astrocytes and microglia in vitro. In astrocytes, OSM-induced STAT1 phosphorylation was significantly greater than for hyperIL-6 (IL-6 linked to the soluble IL-6 receptor) or LIF. However, STAT3 phosphorylation was induced to similar levels for all three cytokines. Conversely, in microglia which lacked the OSMR, hyperIL-6 and LIF but not OSM, induced phosphorylation of STAT3 and only minimal phosphorylation of STAT1. In STAT1 KO astrocytes, OSM conferred a stronger activation of STAT3 compared with WT astrocytes. Transcriptomic analysis of WT and STAT1 KO astrocyte response to OSM, hyperIL-6 or LIF revealed up-regulation and downregulation of many transcripts with OSM > IL-6 > LIF. A dominant IFN-like response to OSM was largely abolished in STAT1 KO astrocytes, suggesting that a large portion of OSM-regulated genes is STAT1-dependent. The gene expression profile of OSM- and hyperIL-6 treated astrocytes was somewhat similar while that for LIF differed the most. Despite signalling via common gp130 pathways, our findings show that specificity is achieved through multiple mechanisms including cell-restricted expression of some receptors, varying strength of signal pathway activation and differential activation of STAT1 and STAT3, which mediate differential gene expression and potential functions by the gp130 cytokines in astrocytes and microglia.

Nonbiased Molecular Screening Identifies Novel Molecular Regulators of Fibrogenic and Proliferative Signaling in Myxomatous Mitral Valve Disease.

Pathological processes underlying myxomatous mitral valve degeneration (MMVD) remain poorly understood. We sought to identify novel mechanisms contributing to the development of this condition. Microarrays were used to measure gene expression in 11 myxomatous and 11 nonmyxomatous human mitral valves. Differential gene expression (thresholds P<0.05; fold-change >1.5) and pathway activation (Ingenuity) were confirmed using quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Contributions of bone morphogenetic protein 4 and transforming growth factor (TGF)-β2 to differential gene expression were evaluated in vitro. Contributions of angiotensin II to differential pathway activation were examined in mice in vivo. A total of 2602 genes were differentially expressed between myxomatous and nonmyxomatous valves. Canonical TGF-β signaling was increased in MMVD because of increased ligand expression and derepression of SMA mothers against decapentaplegic 2/3 signaling and was confirmed with quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Myxomatous valves demonstrated activation of canonical bone morphogenetic protein and Wnt/β-catenin signaling and upregulation of their common target runt-related transcription factor 2. Our data set provided transcriptional and immunohistochemical evidence for activated immune cell infiltration. In vitro treatment of mitral valve interstitial cells with TGF-β2 increased β-catenin signaling at mRNA and protein levels, suggesting interactions between TGF-β2 and Wnt signaling. In vivo infusion of mice with angiotensin II recaptured several changes in signaling pathways characteristic of human MMVD. These data support a new disease framework whereby activation of TGF-β2, bone morphogenetic protein 4, Wnt/β-catenin, or immune signaling plays major roles in the pathogenesis of MMVD. We propose these pathways act in a context-dependent manner to drive phenotypic changes that fundamentally differ from those observed in aortic valve disease and open novel avenues guiding future research into the pathogenesis of MMVD.