Unique Gene Expression Patterns Research Articles

55P Molecular risk factors for distant metastases in premenopausal patients with HR+/HER2- EBC

Breast cancer ranks first in females, concerning cancer incidence and mortality. Breast cancer in premenopausal patients (vs. postmenopausal patients) has unique gene expression patterns, rendering molecular drivers of tumor progression in premenopausal women of particular clinical interest. Our research focused on premenopausal patients with hormone receptor-positive (HR+), human epidermal growth factor receptor 2 negative (HER2-), early breast cancer (EBC), and the molecular drivers of distant metastases after standard adjuvant treatment.

MED12-Related (Neuro)Developmental Disorders: A Question of Causality.

MED12 is a member of the Mediator complex that is involved in the regulation of transcription. Missense variants in MED12 cause FG syndrome, Lujan-Fryns syndrome, and Ohdo syndrome, as well as non-syndromic intellectual disability (ID) in hemizygous males. Recently, female patients with de novo missense variants and de novo protein truncating variants in MED12 were described, resulting in a clinical spectrum centered around ID and Hardikar syndrome without ID. The missense variants are found throughout MED12, whether they are inherited in hemizygous males or de novo in females. They can result in syndromic or nonsyndromic ID. The de novo nonsense variants resulting in Hardikar syndrome that is characterized by facial clefting, pigmentary retinopathy, biliary anomalies, and intestinal malrotation, are found more N-terminally, whereas the more C-terminally positioned variants are de novo protein truncating variants that cause a severe, syndromic phenotype consisting of ID, facial dysmorphism, short stature, skeletal abnormalities, feeding difficulties, and variable other abnormalities. This broad range of distinct phenotypes calls for a method to distinguish between pathogenic and non-pathogenic variants in MED12. We propose an isogenic iNeuron model to establish the unique gene expression patterns that are associated with the specific MED12 variants. The discovery of these patterns would help in future diagnostics and determine the causality of the MED12 variants.

LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA.

Quorum sensing (QS) is a process of chemical communication bacteria use to transition between individual and collective behaviors. QS depends on the production, release, and synchronous response to signaling molecules called autoinducers (AIs). The marine bacterium Vibrio harveyi monitors AIs using a signal transduction pathway that relies on five small regulatory RNAs (called Qrr1-5) that post-transcriptionally control target genes. Curiously, the small RNAs largely function redundantly making it difficult to understand the necessity for five of them. Here, we identify LuxT as a transcriptional repressor of qrr1. LuxT does not regulate qrr2-5, demonstrating that qrr genes can be independently controlled to drive unique downstream QS gene expression patterns. LuxT reinforces its control over the same genes it regulates indirectly via repression of qrr1, through a second transcriptional control mechanism. Genes dually regulated by LuxT specify public goods including an aerolysin-type pore-forming toxin. Phylogenetic analyses reveal that LuxT is conserved among Vibrionaceae and sequence comparisons predict that LuxT represses qrr1 in additional species. The present findings reveal that the QS regulatory RNAs can carry out both shared and unique functions to endow bacteria with plasticity in their output behaviors.

Characterization of IPSCs-Derived Cardiomyocytes

Purpose Functional cardiomyocytes are instrumental to in vitro studies of myocardial diseases, understanding toxicities, and studying new therapies. Because functional cardiomyocytes in tissue culture are scarce and difficult to obtain we sought to differentiate and characterize cardiomyocytes derived from induced pluripotent stem cells (iPSCs) in our lab. Methods Induced pluripotent stem cells (iPSCs) from dermal fibroblasts obtained from a healthy donor were utilized to derive cardiomyocytes. Cells were cultured until 70-85% confluent and differentiated to cardiomyocytes using differentiation kit that contain a high dose of GSK3B inhibitor to induce mesodermal lineage commitment for one day that will result in up-regulation of endogenous BMP4 and activin/NODAL followed by WNT signaling modulation for two days for Cardiac mesodermal induction. Immunocytochemistry for NKX2-5 and TNNT2 were used to characterize differentiated cardiomyocytes. NanoString micro RNA was used to profile commercial and lab differentiated cardiomyocytes. Results Cells start beating on day eight and stayed beating for 30 days in maintenance media. Furthermore, cells were positive for two key markers of the human cardiac lineage: NKX2-5 for early cardiac mesoderm and TNNT2/cTNT for cardiomyocytes. The percentage of differentiation to cardiomyocytes were about 70% of the total iPSCs. Both commercial and lab dedifferentiated cardiomyocytes have similar contractility function. We compared micro RNA profile of our differentiated cardiomyocytes to commercial iPSCs-derived cardiomyocytes and they cluster very close to each other. However, both were clustered differently from miRNA profile of normal adult tissue cardiomyocytes. Conclusion Unlimited iPSCs-derived cardiomyocytes can be obtained in a robust and cost-effective method from fibroblasts. The cells have similar miRNA profile, molecular markers and contractile function in comparison to commercially available cardiomyocytes but have unique patterns of gene expression in comparison to non-failing adult tissue.

Endotype of allergic asthma with airway obstruction in urban children

Black and Hispanic children growing up in disadvantaged urban neighborhoods have the highest rates of asthma and related morbidity in the United States. This study sought to identify specific respiratory phenotypes of health and disease in this population, associations with early life exposures, and molecular patterns of gene expression in nasal epithelial cells that underlie clinical disease. The study population consisted of 442 high-risk urban children who had repeated assessments of wheezing, allergen-specific IgE, and lung function through 10 years of age. Phenotypes were identified by developing temporal trajectories for these data, and then compared to early life exposures and patterns of nasal epithelial gene expression at 11 years of age. Of the 6 identified respiratory phenotypes, a high wheeze, high atopy, low lung function group had the greatest respiratory morbidity. In early life, this group had low exposure to common allergens and high exposure to ergosterol in house dust. While all high-atopy groups were associated with increased expression of a type-2 inflammation gene module in nasal epithelial samples, an epithelium IL-13 response module tracked closely with impaired lung function, and a MUC5AC hypersecretion module was uniquely upregulated in the high wheeze, high atopy, low lung function group. In contrast, a medium wheeze, low atopy group showed altered expression of modules of epithelial integrity, epithelial injury, and antioxidant pathways. In the first decade of life, high-risk urban children develop distinct phenotypes of respiratory health versus disease that link early life environmental exposures to childhood allergic sensitization and asthma. Moreover, unique patterns of airway gene expression demonstrate how specific molecular pathways underlie distinct respiratory phenotypes, including allergic and nonallergic asthma.

Membrane voltage as a dynamic platform for spatiotemporal signaling, physiological, and developmental regulation.

Membrane voltage arises from the transport of ions through ion-translocating ATPases, ion-coupled transport of solutes, and ion channels, and is an integral part of the bioenergetic “currency” of the membrane. The dynamics of membrane voltage—so-called action, systemic, and variation potentials—have also led to a recognition of their contributions to signal transduction, both within cells and across tissues. Here, we review the origins of our understanding of membrane voltage and its place as a central element in regulating transport and signal transmission. We stress the importance of understanding voltage as a common intermediate that acts both as a driving force for transport—an electrical “substrate”—and as a product of charge flux across the membrane, thereby interconnecting all charge-carrying transport across the membrane. The voltage interconnection is vital to signaling via second messengers that rely on ion flux, including cytosolic free Ca2+, H+, and the synthesis of reactive oxygen species generated by integral membrane, respiratory burst oxidases. These characteristics inform on the ways in which long-distance voltage signals and voltage oscillations give rise to unique gene expression patterns and influence physiological, developmental, and adaptive responses such as systemic acquired resistance to pathogens and to insect herbivory.

Abstract PO007: Evaluation of crosstalk between estrogen and Wnt signaling in endometrial cancer

Abstract Estrogen signaling through estrogen receptor alpha (ER) is considered to be a driving oncogenic signal in type 1 endometrial cancer. However, excess estrogen exposure isn’t sufficient to initiate tumorigenesis in mouse models, suggesting additional genomic or epigenomic changes are required for tumor formation. Activating mutations in beta-catenin, a downstream regulator of Wnt signaling, are prevalent in type I endometrial tumors, with almost 40% of cases harboring these mutations. The combination of excess estrogen and Wnt signals appears to drive endometrial cancer formation and growth, but the mechanisms underlying interplay between these pathways is not well understood. Based on previous studies and our preliminary data, we are currently exploring the hypothesis that beta-catenin acts as a cofactor for ER, representing a convergence of these two oncogenic pathways. Using Ishikawa cells, a type 1 endometrial cancer cell line expressing wildtype beta-catenin, we are measuring differences in gene expression and chromatin between cells that have been induced with 17-beta-estradiol (E2), Wnt3a, or the combination. Our findings suggest that a combination induction affects where ER and beta-catenin bind the genome. In the presence of E2 and Wnt3a, ER’s genomic occupancy expanded and revealed Wnt dependent ER sites separate from canonical ER and Wnt binding sites. Analysis of acetylation of lysine 27 on histone H3 (H3K27ac) using ChIP-seq support this pattern, where acetylation is enriched in the combination treatment over an E2 or Wnt3a induction alone. Additionally, we observed unique gene expression patterns with a simultaneous E2 and Wnt3a induction. We are in the process of creating an isogenic Ishikawa cell line that harbors an activating beta-catenin mutation to determine molecular differences between Wnt ligand activation and constitutively active beta-catenin and discover how these forms of Wnt activation relate to estrogen signaling. Overall, we hope our studies deepen our understanding of estrogen driven transcriptional regulation and shed light on the functional consequences of beta-catenin activating mutations in endometrial cancer. Citation Format: Krystle Osby, Adriana C. Rodriguez, Jay Gertz. Evaluation of crosstalk between estrogen and Wnt signaling in endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO007.

Changes in the Immune Phenotype and Gene Expression Profile Driven by a Novel Tuberculosis Nanovaccine: Short and Long-Term Post-immunization.

Deciphering protection mechanisms against Mycobacterium tuberculosis (Mtb) remains a critical challenge for the development of new vaccines and therapies. We analyze the phenotypic and transcriptomic profile in lung of a novel tuberculosis (TB) nanoparticle-based boosting mucosal vaccine Nano-FP1, which combined to BCG priming conferred enhanced protection in mice challenged with low-dose Mtb. We analyzed the vaccine profile and efficacy at short (2 weeks), medium (7 weeks) and long term (11 weeks) post-vaccination, and compared it to ineffective Nano-FP2 vaccine. We observed several changes in the mouse lung environment by both nanovaccines, which are lost shortly after boosting. Additional boosting at long-term (14 weeks) recovered partially cell populations and transcriptomic profile, but not enough to enhance protection to infection. An increase in both total and resident memory CD4 and CD8 T cells, but no pro-inflammatory cytokine levels, were correlated with better protection. A unique gene expression pattern with differentially expressed genes revealed potential pathways associated to the immune defense against Mtb. Our findings provide an insight into the critical immune responses that need to be considered when assessing the effectiveness of a novel TB vaccine.

Manganese exposure in juvenile C57BL/6 mice increases glial inflammatory responses in the substantia nigra following infection with H1N1 influenza virus.

Infection with Influenza A virus can lead to the development of encephalitis and subsequent neurological deficits ranging from headaches to neurodegeneration. Post-encephalitic parkinsonism has been reported in surviving patients of H1N1 infections, but not all cases of encephalitic H1N1 infection present with these neurological symptoms, suggesting that interactions with an environmental neurotoxin could promote more severe neurological damage. The heavy metal, manganese (Mn), is a potential interacting factor with H1N1 because excessive exposure early in life can induce long-lasting effects on neurological function through inflammatory activation of glial cells. In the current study, we used a two-hit model of neurotoxin-pathogen exposure to examine whether exposure to Mn during juvenile development would induce a more severe neuropathological response following infection with H1N1 in adulthood. To test this hypothesis, C57BL/6 mice were exposed to MnCl2 in drinking water (50 mg/kg/day) for 30 days from days 21-51 postnatal, then infected intranasally with H1N1 three weeks later. Analyses of dopaminergic neurons, microglia and astrocytes in basal ganglia indicated that although there was no significant loss of dopaminergic neurons within the substantia nigra pars compacta, there was more pronounced activation of microglia and astrocytes in animals sequentially exposed to Mn and H1N1, as well as altered patterns of histone acetylation. Whole transcriptome Next Generation Sequencing (RNASeq) analysis was performed on the substantia nigra and revealed unique patterns of gene expression in the dual-exposed group, including genes involved in antioxidant activation, mitophagy and neurodegeneration. Taken together, these results suggest that exposure to elevated levels of Mn during juvenile development could sensitize glial cells to more severe neuro-immune responses to influenza infection later in life through persistent epigenetic changes.

Modeling the effects of respirasome supercomplex formation on AD progression by incorporation of mitochondrial gene expression data

AbstractBackgroundAlzheimer’s disease is a specific form of dementia characterized by the aggregation of amyloid‐β plaques and tau tangles. New research has found that the formation of these aggregates occurs after dysregulation of respiratory chain activity. Proteomic and genomic data show that these changes are also related to unique gene expression patterns. Supercomplexes (SCs) are assemblages of multiple, functional respiratory complexes. After individual complexes have formed, respiratory complexes (RC) I, III, and IV begin to associate and form SCs. SCs are stable intermediates between respirasomes and individual complexes. We investigated the impact of our observed gene expression changes on new therapeutic options via metabolic flux analysis of SC assembly.MethodTotal RNA was isolated from 80‐180 mg of frozen brain tissue from 11 AD and 5 control samples. Total RNA was quantified and assessed for purity using Nanodrop and Bioanalyzer analysis. cDNA was prepared and applied to the Human Mitochondrial Energy Metabolism and the Mitochondria RT² Profiler™ PCR Arrays. The qPCR data was then analyzed using Qiagen RT2 Profiler PCR Array Data Analysis version 3.5. Gene expression changes were incorporated into the mathematical model created in CellDesigner 4.2 and these were converted into a series of mathematical equations by SBMLsqueezer. This file was imported into COPASI, a biological pathway simulation program. Treatment data were found using COPASI’s time course task. Each treatment represented a mathematical change in gene expression of a selected enzyme.ResultWe investigated 9 mitochondrial genes known to be important for efficient SC assembly. In all cases, the concentration of the gene products was decreased in the AD brain samples. After simulation of respirasome assembly using a reduced model only UQCRC1 treatment increased the percent of CI sequestration into respirasomes.ConclusionOur results indicate that protective changes in expression of UQCRCI, a component of electron transport chain respiratory complex III may serve as a potential avenue for AD therapeutics. This work also highlights the importance of metabolic enzyme activity in maintaining proper respiratory activity.We would like to acknowledge support for this work from the Alzheimer’s and Related Diseases Research Award Fund and the Commonwealth Health Research Board.

Alteration of tumor-associated macrophage subtypes mediated by KRT6A in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is severely affecting the health and lives of patients. Clarifying the composition and regulatory factors of tumor immune microenvironment (TIME) is helpful for the treatment of PDAC. We analyzed the unique TIMEs and gene expression patterns between PDAC and adjacent normal tissue (ANT) using Gene Expression Omnibus (GEO) to find new immunotherapy targets. The Cancer Genome Atlas (TCGA) datasets were used to elucidate the possible mechanism of which tumor-associated macrophages (TAMs) changed in PDAC. We found that the composition of TAMs subtypes, including M0, M1, and M2, was different between PDAC and ANT, which was validated in recently published single-cell RNA-seq data. Many immune cells interacted with each other to affect the TIME. There were many DEGs enriched in some pathways that could potentially change the immune cell composition. KRT6A was found to be a DEG between PDAC and ANT that overlapped with DEGs between the M0-high group and the M0-low group in TCGA datasets, and it might alter and regulate TAMs via a collection of genes including COL5A2, COL1A2, MIR3606, SPARC, and COL6A3. TAMs, which could be a target of immunotherapy, might be influenced by genes through KRT6A and indicate an undesirable prognosis in PDAC.

Hypoxia Alters the Response to Anti-EGFR Therapy by Regulating EGFR Expression and Downstream Signaling in a DNA Methylation–Specific and HIF-Dependent Manner

Intratumoral hypoxia occurs in 90% of solid tumors and is associated with a poor prognosis for patients. Cancer cells respond to hypoxic microenvironments by activating the transcription factors, hypoxia-inducible factor 1 (HIF1) and HIF2. Here, we studied the unique gene expression patterns of 31 different breast cancer cell lines exposed to hypoxic conditions. The EGFR, a member of the ErbB (avian erythroblastosis oncogene B) family of receptors that play a role in cell proliferation, invasion, metastasis, and apoptosis, was induced in seven of the 31 breast cancer cell lines by hypoxia. A functional hypoxia response element (HRE) was identified, which is activated upon HIF1 binding to intron 18 of the EGFR gene in cell lines in which EGFR was induced by hypoxia. CpG methylation of the EGFR HRE prevented induction under hypoxic conditions. The HRE of EGFR was methylated in normal breast tissue and some breast cancer cell lines, and could be reversed by treatment with DNA methyltransferase inhibitors. Induction of EGFR under hypoxia led to an increase in AKT, ERK, and Rb phosphorylation as well as increased levels of cyclin D1, A, B1, and E2F, and repression of p21 in an HIF1α-dependent manner, leading to cell proliferation and migration. Also, increased EGFR expression sensitized cells to EGFR inhibitors. Collectively, our data suggest that patients with hypoxic breast tumors and hypomethylated EGFR status may benefit from EGFR inhibitors currently used in the clinic. SIGNIFICANCE: Hypoxia sensitizes breast cancer cells to EGFR inhibitors in an HIF1α- and a methylation-specific manner, suggesting patients with hypoxic tumors may benefit from EGFR inhibitors already available in the clinic. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/22/4998/F1.large.jpg.

Exploration into biomarker potential of region-specific brain gene co-expression networks

The human brain is a complex organ that consists of several regions each with a unique gene expression pattern. Our intent in this study was to construct a gene co-expression network (GCN) for the normal brain using RNA expression profiles from the Genotype-Tissue Expression (GTEx) project. The brain GCN contains gene correlation relationships that are broadly present in the brain or specific to thirteen brain regions, which we later combined into six overarching brain mini-GCNs based on the brain’s structure. Using the expression profiles of brain region-specific GCN edges, we determined how well the brain region samples could be discriminated from each other, visually with t-SNE plots or quantitatively with the Gene Oracle deep learning classifier. Next, we tested these gene sets on their relevance to human tumors of brain and non-brain origin. Interestingly, we found that genes in the six brain mini-GCNs showed markedly higher mutation rates in tumors relative to matched sets of random genes. Further, we found that cortex genes subdivided Head and Neck Squamous Cell Carcinoma (HNSC) tumors and Pheochromocytoma and Paraganglioma (PCPG) tumors into distinct groups. The brain GCN and mini-GCNs are useful resources for the classification of brain regions and identification of biomarker genes for brain related phenotypes.

Function and transcriptomic dynamics of Sertoli cells during prospermatogonia development in mouse testis

In mammals, spermatogonial stem cells (SSCs) arise from a subpopulation of prospermatogonia during neonatal testis development. Currently, molecular mechanisms directing the prospermatogonia to spermatogonial transition are not well understood. In the study, we found that reducing Sertoli cells number by Amh-cre mediated expression of diphtheria toxin (AC;DTA) in murine fetal testis caused defects in prospermatogonia fate decisions. Histological and immunohistochemical analyses confirmed that Sertoli cells loss occurred at embryonic day (E) 14.5. Prospermatogonia maintained mitotic arrest at E16.5 in control animals, in contrast, 13.4% of germ cells in AC;DTA testis reentered cell cycle and expressed gH2A.X and Sycp3, indicating the commitment to meiosis. After birth, the number of prospermatogonia resuming mitosis was significantly affected by Sertoli cell loss in AC;DTA animals. Lastly, we isolated primary Sertoli cells using a Sertoli cell specific GFP reporter line and showed dynamics of Sertoli cell transcriptomes at E12.5, E13.5, E16.5 and P1. By further analysis, we revealed unique gene expression patterns and potential candidate genes regulating Sertoli cell development and likely mediating interactions between Sertoli cells, prospermatogonia and other testicular cells.

Abstract 232: Comprehensive metabolic profiling and vulnerabilities to metabolic inhibitors among small cell lung cancer subtypes

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor that constitutes approximately 14% of all lung cancers diagnosed in the United States. Despite recent advances in treatment options, recurrence is a major challenge and long-term survival remains poor underscoring the importance of investigating mechanisms of treatment resistance and therapeutic strategies. Interestingly, our laboratory has recently uncovered evidence for the classification of SCLC into distinct subtypes, each with unique gene expression patterns and therapeutic vulnerabilities. These subtypes are characterized by differential expression of the transcription factors ASCL1, NEUROD1, or POU2F3. A fourth subtype is negative for all three transcription factors and has prominent expression of immune genes, thus is termed the Inflamed subtype. These subtypes are associated with differences in drug sensitivity, biomarker signatures, and mechanisms of cellular growth. One facet of tumor biology that has not been explored across the subtypes, or in SCLC in general, is the metabolic mechanisms of energy generation. Although metabolic reprogramming from mitochondrial respiration to glycolysis is considered a major hallmark of cancer, increasing evidence suggests that mitochondrial or amino acid dependencies may also contribute to treatment resistance. Therefore, understanding the metabolic demand, particularly among the subtypes, is critical for the development of new therapies. To address this, gene set enrichment analysis was applied to 81 human SCLC tumors categorized by subtype, and revealed differential gene expression correlating to pathways such as fatty acid metabolism in POU2F3 and reactive oxygen species in Inflamed that was confirmed by reverse phase protein analysis (RPPA). Further, we evaluated nutrient requirements, glycolysis dependence, and mitochondrial function in human SCLC cell lines and found differences in glucose uptake and subsequent lactate generation between the subtypes suggesting that glycolysis inhibition may be effective in particular subsets. Based on this, we then tested the efficacy of PFK-158 (glycolysis inhibitor) on cellular proliferation and metabolic function in these cell lines, which revealed variations in susceptibility dependent on subtype classification. Together, this data serves to enhance the current understanding of SCLC mechanisms of growth and provides evidence for metabolic inhibition as a potential therapeutic opportunity. Citation Format: Kasey R. Cargill, Carl M. Gay, Robert J. Cardnell, You-Hong Fan, Qi Wang, Lixia Diao, Jing Wang, Lauren A. Byers. Comprehensive metabolic profiling and vulnerabilities to metabolic inhibitors among small cell lung cancer subtypes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 232.

Abstract 4441: CS3003, an HDAC6-selective inhibitor, improves anti-PD-1 immune checkpoint blockade therapy efficacy

Abstract Background: Histone deacetylases (HDACs) are involved in diverse cellular regulatory function including histone modification and non-histone modification. Several publications have demonstrated that selective HDAC inhibitors (HDACi) can influence tumor growth and immunogenicity. In particular, the selective inhibition of HDAC6 has function of inhibiting tumor growth in several malignancies. However, the mechanism of this effect is still not clear. Methods: In this study, we evaluated CS3003, a selective HDAC6 inhibitor, inhibition profile using cell-based free assays. In vivo anti-tumor efficacy was evaluated in MM.1S xenograft mouse model. CS3003 and PD-1 blockade combination effect was evaluated in CT26 syngeneic mouse model. RNA-seq method was used to explore the transcription regulation by CS3003 compared to other pan-HDAC, Class I HDAC inhibitor and selective HDAC6 inhibitors. Results: CS3003 has distinct inhibition profile in HDAC family members. Combination of CS3003 and proteasome inhibitor synergistically enhanced anti-tumor function in human multiple myeloma mouse model (coefficient of drug interaction, CDI=0.69). In addition, CS3003 improves the anti-tumor activity of anti-PD-1 immune checkpoint blockade in solid tumor model (CDI=0.62 and 0.88 when the dosage of CS3003 was 30 and 70mg/kg, respectively). Transcriptome analysis demonstrated a unique gene expression pattern of CS3003 from other pan-HDAC and selective HDAC6 inhibitors. Conclusions: These data provide a comprehensive pre-clinical characterization of CS3003 that supports its unique HDAC inhibition profile and its capability of improving anti-PD-1 immune checkpoint blockade therapy efficacy. Citation Format: Zhenhu Li, Zhaoxiang Ren, Jingshu Ma, Liang Tang, Liang Lu, Ying Zhu, Yunfei Wu, Tiancheng Liu, Juan Zhang, Yuanwu Bao, Kangyu Zhang, Changqing Wei, Shuhui Chen, Xinzhong Jon Wang. CS3003, an HDAC6-selective inhibitor, improves anti-PD-1 immune checkpoint blockade therapy efficacy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4441.

An overview of neuroblastoma cell lineage phenotypes and in vitro models.

This review was conducted to present the main neuroblastoma (NB) clinical characteristics and the most common genetic alterations present in these pediatric tumors, highlighting their impact in tumor cell aggressiveness behavior, including metastatic development and treatment resistance, and patients' prognosis. The distinct three NB cell lineage phenotypes, S-type, N-type, and I-type, which are characterized by unique cell surface markers and gene expression patterns, are also reviewed. Finally, an overview of the most used NB cell lines currently available for in vitro studies and their unique cellular and molecular characteristics, which should be taken into account for the selection of the most appropriate model for NB pre-clinical studies, is presented. These valuable models can be complemented by the generation of NB reprogrammed tumor cells or organoids, derived directly from patients' tumor specimens, in the direction toward personalized medicine.

Quantitative gene expression dynamics of key placode signalling factors in the embryonic chicken scleral ossicle system

Development of the scleral ossicles, a ring of bony elements within the sclera, is directed by a series of papillae that arise from placodes in the conjunctival epithelium over a 1.5-day induction period in the chicken embryo. The regular spacing of the papillae around the corneal-scleral limbus suggests that their induction may be regulated by a reaction-diffusion mechanism, similar to other epithelial appendages. Some key placode signalling molecules, including β-catenin, are known to be expressed throughout the induction period. However, others have been studied only at certain stages or have not been successfully detected. Here we use qPCR to study the gene expression patterns of the wingless integration (WNT)/β-catenin, bone morphogenetic protein (BMP), ectodysplasin (EDA), fibroblast growth factor (FGF) and hedgehog (HH) signalling families in discrete regions of the eye throughout the complete conjunctival placode and papillae induction period. This comprehensive analysis revealed a variable level of gene expression within specific eye regions, with some genes exhibiting high, moderate or low changes. Most genes exhibited an initial increase in gene expression, followed by a decrease or plateau as development proceeded, suggesting that some genes are important for a brief initial period whilst the sustained elevated expression level of other genes is needed for developmental progression. The timing or magnitude of these changes, and/or the overall gene expression trend differed in the temporal, nasal and/or dorsal eye regions for some, but not all genes, demonstrating that gene expression may vary across different eye regions. Temporal and nasal EDA receptor (EDAR) had the greatest number of strong correlations (r > 0.700) with other genes and β-catenin had the greatest number of moderate correlations (r = 0.400–0.700), while EDA had the greatest range in correlation strengths. Among the strongly correlated genes, two distinct signalling modules were identified, connected by some intermediate genes. The dynamic gene expression patterns of the five signalling pathways studied here from conjunctival placode formation through to papillae development is consistent with other epithelial appendages and confirms the presence of a conserved induction and patterning signalling network. Two unique gene expression patterns and corresponding gene interaction modules suggest functionally distinct roles throughout placode development. Furthermore, spatial differences in gene expression patterns among the temporal, nasal and dorsal regions of the eye may indicate that the expression of certain genes is influenced by mechanical forces exerted throughout development. Therefore, this study identifies key placode signalling factors and their interactions, as well as some potential region-specific features of gene expression in the scleral ossicle system and provides a basis for further exploration of the spatial expression of these genes and the patterning mechanism(s) active throughout conjunctival placode and papillae formation.

Comparative transcriptome analysis of two Cercospora sojina strains reveals differences in virulence under nitrogen starvation stress

BackgroundCercospora sojina is a fungal pathogen that causes frogeye leaf spot in soybean-producing regions, leading to severe yield losses worldwide. It exhibits variations in virulence due to race differentiation between strains. However, the candidate virulence-related genes are unknown because the infection process is slow, making it difficult to collect transcriptome samples.ResultsIn this study, virulence-related differentially expressed genes (DEGs) were obtained from the highly virulent Race 15 strain and mildly virulent Race1 strain under nitrogen starvation stress, which mimics the physiology of the pathogen during infection. Weighted gene co-expression network analysis (WGCNA) was then used to find co-expressed gene modules and assess the relationship between gene networks and phenotypes. Upon comparison of the transcriptomic differences in virulence between the strains, a total of 378 and 124 DEGs were upregulated, while 294 and 220 were downregulated in Race 1 and Race 15, respectively. Annotation of these DEGs revealed that many were associated with virulence differences, including scytalone dehydratase, 1,3,8-trihydroxynaphthalene reductase, and β-1,3-glucanase. In addition, two modules highly correlated with the highly virulent strain Race 15 and 36 virulence-related DEGs were found to contain mostly β-1,4-glucanase, β-1,4-xylanas, and cellobiose dehydrogenase.ConclusionsThese important nitrogen starvation-responsive DEGs are frequently involved in the synthesis of melanin, polyphosphate storage in the vacuole, lignocellulose degradation, and cellulose degradation during fungal development and differentiation. Transcriptome analysis indicated unique gene expression patterns, providing further insight into pathogenesis.

Brain regional gene expression network analysis identifies unique interactions between chronic ethanol exposure and consumption.

Progressive increases in ethanol consumption is a hallmark of alcohol use disorder (AUD). Persistent changes in brain gene expression are hypothesized to underlie the altered neural signaling producing abusive consumption in AUD. To identify brain regional gene expression networks contributing to progressive ethanol consumption, we performed microarray and scale-free network analysis of expression responses in a C57BL/6J mouse model utilizing chronic intermittent ethanol by vapor chamber (CIE) in combination with limited access oral ethanol consumption. This model has previously been shown to produce long-lasting increased ethanol consumption, particularly when combining oral ethanol access with repeated cycles of intermittent vapor exposure. The interaction of CIE and oral consumption was studied by expression profiling and network analysis in medial prefrontal cortex, nucleus accumbens, hippocampus, bed nucleus of the stria terminalis, and central nucleus of the amygdala. Brain region expression networks were analyzed for ethanol-responsive gene expression, correlation with ethanol consumption and functional content using extensive bioinformatics studies. In all brain-regions studied the largest number of changes in gene expression were seen when comparing ethanol naïve mice to those exposed to CIE and drinking. In the prefrontal cortex, however, unique patterns of gene expression were seen compared to other brain-regions. Network analysis identified modules of co-expressed genes in all brain regions. The prefrontal cortex and nucleus accumbens showed the greatest number of modules with significant correlation to drinking behavior. Across brain-regions, however, many modules with strong correlations to drinking, both baseline intake and amount consumed after CIE, showed functional enrichment for synaptic transmission and synaptic plasticity.