Premature aging and reduced cancer incidence associated with near-complete body-wide Myc inactivation.

MYC proto-oncogene dysregulation alters metabolism, translation, and other functions in ways that support tumor induction and maintenance. Although Myc+/- mice are healthier and longer-lived than control mice, the long-term ramifications of more complete Myc loss remain unknown. We now describe the chronic consequences of body-wide Myc inactivation initiated postnatally. "MycKO" mice acquire numerous features of premature aging, including altered body composition and habitus, metabolic dysfunction, hepatic steatosis, and dysregulation of gene sets involved in functions that normally deteriorate with aging. Yet, MycKO mice have extended lifespans that correlate with a 3- to 4-fold lower lifetime cancer incidence. Aging tissues from normal mice and humans also downregulate Myc and gradually alter many of the same Myc target gene sets seen in MycKO mice. Normal aging and its associated cancer predisposition are thus highly linked via Myc.

Differential gene expression in peripheral blood mononuclear cells from children immunized with inactivated influenza vaccine.

The human immune response to inactivated influenza vaccine is dynamic and impacted by age and preexisting immunity. Our goal was to identify postvaccination transcriptomic changes in peripheral blood mononuclear cells from children. Blood samples were obtained before and at 3 or 7 days postvaccination with 2016-2017 quadrivalent inactivated influenza vaccine and RNA sequencing was performed. There were 1,466 differentially expressed genes (DEGs) for the Day 0-Day 3 group and 513 DEGs for the Day 0-Day 7 group. Thirty-three genes were common between the two groups. The majority of the transcriptomic changes at Day 3 represented innate inflammation and apoptosis pathways. Day 7 DEGs were characterized by activation of cellular processes, including the regulation of cytoskeleton, junctions, and metabolism, and increased expression of immunoglobulin genes. DEGs at Day 3 were compared between older and younger children revealing increased inflammatory gene expression in the older group. Vaccine history in the year prior to the study was characterized by robust DEGs at Day 3 with decreased phagosome and dendritic cell maturation in those who had been vaccinated in the previous year. PBMC responses to inactivated influenza vaccination in children differed significantly by the timing of sampling, patient age, and vaccine history. These data provide insight into the expected molecular pathways to be temporally altered by influenza vaccination in children.

Sequential analysis of transcript expression patterns improves survival prediction in multiple cancers.

BACKGROUND: Long-term survival in numerous cancers often correlates with specific whole transcriptome profiles or the expression patterns of smaller numbers of transcripts. In some instances, these are better predictors of survival than are standard classification methods such as clinical stage or hormone receptor status in breast cancer. Here, we have used the method of "t-distributed stochastic neighbor embedding" (t-SNE) to show that, collectively, the expression patterns of small numbers of functionally-related transcripts from fifteen cancer pathways correlate with long-term survival in the vast majority of tumor types from The Cancer Genome Atlas (TCGA). We then ask whether the sequential application of t-SNE using the transcripts from a second pathway improves predictive capability or whether t-SNE can be used to refine the initial predictive power of whole transcriptome profiling. METHODS: RNAseq data from 10,227 tumors in TCGA were previously analyzed using t-SNE-based clustering of 362 transcripts comprising 15 distinct cancer-related pathways. After showing that certain clusters were associated with differential survival, each relevant cluster was re-analyzed by t-SNE with a second pathway's transcripts. Alternatively, groups with differential survival identified by whole transcriptome profiling were subject to a second, t-SNE-based analysis. RESULTS: Sequential analyses employing either t-SNE➔t-SNE or whole transcriptome profiling➔t-SNE analyses were in many cases superior to either individual method at predicting long-term survival. We developed a dynamic and intuitive R Shiny web application to explore the t-SNE based transcriptome clustering and survival analysis across all TCGA cancers and all 15 cancer-related pathways in this analysis. This application provides a simple interface to select specific t-SNE clusters and analyze survival predictability using both individual or sequential approaches. The user can recreate the relationships described in this analysis and further explore many different cancer, pathway, and cluster combinations. Non-R users can access the application on the web at https://chpupsom19.shinyapps.io/Survival_Analysis_tsne_umap_TCGA. The application, R scripts performing survival analysis, and t-SNE clustering results of TCGA expression data can be accessed on GitHub enabling users to download and run the application locally with ease (https://github.com/RavulaPitt/Sequential-t-SNE/). CONCLUSIONS: The long-term survival of patients correlated with expression patterns of 362 transcripts from 15 cancer-related pathways. In numerous cases, however, survival could be further improved when the cohorts were re-analyzed using iterative t-SNE clustering or when t-SNE clustering was applied to cohorts initially segregated by whole transcriptome-based hierarchical clustering.

A Novel Small Molecule Targets Androgen Receptor and Its Splice Variants in Castration-Resistant Prostate Cancer.

Reactivation of androgen receptor (AR) appears to be the major mechanism driving the resistance of castration-resistant prostate cancer (CRPC) to second-generation antiandrogens and involves AR overexpression, AR mutation, and/or expression of AR splice variants lacking ligand-binding domain. There is a need for novel small molecules targeting AR, particularly those also targeting AR splice variants such as ARv7. A high-throughput/high-content screen was previously reported that led to the discovery of a novel lead compound, 2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)-1-(4-(2,3-dimethylphenyl)piperazin-1-yl)ethan-1-one (IMTPPE), capable of inhibiting nuclear AR level and activity in CRPC cells, including those resistant to enzalutamide. A novel analogue of IMTPPE, JJ-450, has been investigated with evidence for its direct and specific inhibition of AR transcriptional activity via a pulldown assay and RNA-sequencing analysis, PSA-based luciferase, qPCR, and chromatin immunoprecipitation assays, and xenograft tumor model 22Rv1. JJ-450 blocks AR recruitment to androgen-responsive elements and suppresses AR target gene expression. JJ-450 also inhibits ARv7 transcriptional activity and its target gene expression. Importantly, JJ-450 suppresses the growth of CRPC tumor xenografts, including ARv7-expressing 22Rv1. Collectively, these findings suggest JJ-450 represents a new class of AR antagonists with therapeutic potential for CRPC, including those resistant to enzalutamide.

Inflammatory Mediator Expression Associated With Antibody Response Induced by Live Attenuated vs Inactivated Influenza Virus Vaccine in Children.

BACKGROUND: The reasons for differences in vaccine effectiveness between live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV) are not clear. METHODS: Blood samples were obtained before vaccination and at days 7 and 21 postvaccination with 2015-2016 quadrivalent IIV or LAIV. Serologic response to the vaccine was measured by hemagglutination inhibition assay. Targeted RNA sequencing and serum cytokine analysis were performed. Paired analyses were used to determine gene expression and were compared between IIV and LAIV recipients. Classification And Regression Trees analysis (CART) identified the strongest associations with vaccine response. RESULTS: Forty-six enrollees received IIV, and 25 received LAIV. The mean age was 11.5 (±3.7) years. Seroconversion with IIV was associated with changes in expression of PRKRA and IFI6. Nonseroconversion for both IIV and LAIV was characterized by increased interferon-stimulated gene expression. Seroprotection with both vaccines was associated with altered expression of CXCL2 and CD36. For LAIV, CART showed that changes in expression of CD80, CXCL2, and CASP1 were associated with seroprotection. Serum cytokines showed that IIV seroconversion was associated with decreased CCL3. LAIV seroprotection tracked with decreased tumor necrosis factor-α and interferon-γ. CONCLUSIONS: Distinct markers of seroconversion and seroprotection against IIV and LAIV were identified using immunophenotyping and CART analysis.

The transcriptome of the Didelphis virginiana opossum kidney OK proximal tubule cell line.

The OK cell line derived from the kidney of a female opossum Didelphis virginiana has proven to be a useful model in which to investigate the unique regulation of ion transport and membrane trafficking mechanisms in the proximal tubule (PT). Sequence data and comparison of the transcriptome of this cell line to eutherian mammal PTs would further broaden the utility of this culture model. However, the genomic sequence for D. virginiana is not available and although a draft genome sequence for the opossum Monodelphis domestica (sequenced in 2012 by the Broad Institute) exists, transcripts sequenced from both species show significant divergence. The M. domestica sequence is not highly annotated, and the majority of transcripts are predicted rather than experimentally validated. Using deep RNA sequencing of the D. virginiana OK cell line, we characterized its transcriptome via de novo transcriptome assembly and alignment to the M. domestica genome. The quality of the de novo assembled transcriptome was assessed by the extent of homology to sequences in nucleotide and protein databases. Gene expression levels in the OK cell line, from both the de novo transcriptome and genes aligned to the M. domestica genome, were compared with publicly available rat kidney nephron segment expression data. Our studies demonstrate the expression in OK cells of numerous PT-specific ion transporters and other key proteins relevant for rodent and human PT function. Additionally, the sequence and expression data reported here provide an important resource for genetic manipulation and other studies on PT cell function using these cells.

Immuno-proteomics: Development of a novel reagent for separating antibodies from their target proteins.

Immunoprecipitation (IP) is a widely used technique for identifying the binding partners of the target proteins of specific antibodies. Putative binding targets and their partners are usually in much lower amounts than the antibodies used to capture these target proteins. Thus antigen identification using proteomics following IP is often confounded by the presence of an overwhelming amount of interfering antibody protein. Even covalently linking antibodies to beads is susceptible to antibody leaching during IP. To circumvent this interference, we describe here a reagent, called Biotin-CDM that reversibly tags all potential target proteins in a cell lysate with biotin. The presence of biotin coupled to the target proteins allows for a secondary separation step in which antibodies are washed away from the reversibly biotinylated target proteins by binding them to an Avidin-coupled matrix. The captured target proteins are released from the Avidin matrix by reversing the Biotin-CDM link, thus releasing a pool of target proteins ready for further proteomic analysis compatible with 2D-electrophoresis. Here, we describe the synthesis and characterization of Biotin-CDM. We also demonstrate Biotin-CDM's use for immunoprecipitation of a known antigen, as well as its use for capturing an array of proteins targeted by the autoantibodies found in the serum a patient suffering from rheumatoid arthritis. The use of this reagent allows one to combine immunoprecipitation and 2D-Difference gel electrophoresis, overcoming the current limitations of Serological Proteome Analysis (SERPA) in discovering autoantigens. This article is part of a Special Issue entitled: Medical Proteomics.