Integrated systems for exosome investigation.

Extracellular vesicles, including exosomes, are currently being investigated to better understand their biogenesis and biological functions. There is also a rapidly growing interest in utilizing exosomes present in patient biofluids for molecular diagnostics in the clinic. Exosomes are natural shuttles of RNA and protein cargo, making them attractive as potential therapeutic delivery vehicles. Here, we describe the methods for using the latest tools and technologies to study exosomes to better understand their roles in cell-to-cell communication, for discovery of clinical biomarkers and to engineer exosomes for therapeutic applications.

Intratumoral plasma cells predict outcomes to PD-L1 blockade in non-small cell lung cancer.

Inhibitors of the programmed cell death-1 (PD-1/PD-L1) signaling axis are approved to treat non-small cell lung cancer (NSCLC) patients, based on their significant overall survival (OS) benefit. Using transcriptomic analysis of 891 NSCLC tumors from patients treated with either the PD-L1 inhibitor atezolizumab or chemotherapy from two large randomized clinical trials, we find a significant B cell association with extended OS with PD-L1 blockade, independent of CD8+ T cell signals. We then derive gene signatures corresponding to the dominant B cell subsets present in NSCLC from single-cell RNA sequencing (RNA-seq) data. Importantly, we find increased plasma cell signatures to be predictive of OS in patients treated with atezolizumab, but not chemotherapy. B and plasma cells are also associated with the presence of tertiary lymphoid structures and organized lymphoid aggregates. Our results suggest an important contribution of B and plasma cells to the efficacy of PD-L1 blockade in NSCLC.

Clonal Hematopoiesis in Late-Stage Non-Small-Cell Lung Cancer and Its Impact on Targeted Panel Next-Generation Sequencing.

PURPOSE: Somatic mutations derived from the expansion of clonal populations of blood cells (clonal hematopoiesis of indeterminate potential, or CHIP) may be detected in sequencing of cell-free DNA (cfDNA) samples. We evaluated the potential implications of CHIP in targeted sequencing of plasma samples using matched peripheral blood mononuclear cells (PBMCs) from patients with lung cancer to identify potential CHIP-associated mutations. MATERIALS AND METHODS: A total of 332 plasma and corresponding PBMC samples were collected predose, cycle 1 day 1 (C1D1), from the randomized, phase III study (OAK) comparing atezolizumab versus docetaxel in previously treated patients with non-small-cell lung cancer (NSCLC). The samples were analyzed with the AVENIO ctDNA Surveillance Kit (for research use only; not for use in diagnostic procedures), a 198-kb next-generation sequencing panel targeting cancer-related genes. CHIP variants were assessed by analyzing both plasma and PBMC sequencing data. RESULTS: A range of zero to eight CHIP variants (median = one) was detected per cfDNA sample. Most of these variants were not in the Database of Single Nucleotide Polymorphisms (dbSNP). The number of CHIP variants was positively associated with age, and TP53 was the most frequently mutated gene. Furthermore, the allele frequency was less variable over time for CHIP variants than for tumor-derived variants. CONCLUSION: CHIP-derived mutations are present in late-stage NSCLC. However, not all plasma samples had CHIP mutations detected with targeted panel sequencing. Paired PBMC sequencing analysis may be needed to remove CHIP variants for comprehensive genomic profiling using plasma samples to identify true somatic mutations.

High SINE RNA Expression Correlates with Post-Transcriptional Downregulation of BRCA1.

Short Interspersed Nuclear Elements (SINEs) are non-autonomous retrotransposons that comprise a large fraction of the human genome. SINEs are demethylated in human disease, but whether SINEs become transcriptionally induced and how the resulting transcripts may affect the expression of protein coding genes is unknown. Here, we show that downregulation of the mRNA of the tumor suppressor gene BRCA1 is associated with increased transcription of SINEs and production of sense and antisense SINE small RNAs. We find that BRCA1 mRNA is post-transcriptionally down-regulated in a Dicer and Drosha dependent manner and that expression of a SINE inverted repeat with sequence identity to a BRCA1 intron is sufficient for downregulation of BRCA1 mRNA. These observations suggest that transcriptional activation of SINEs could contribute to a novel mechanism of RNA mediated post-transcriptional silencing of human genes.

Maintenance of interphase chromosome compaction and homolog pairing in Drosophila is regulated by the condensin cap-h2 and its partner Mrg15.

Dynamic regulation of chromosome structure and organization is critical for fundamental cellular processes such as gene expression and chromosome segregation. Condensins are conserved chromosome-associated proteins that regulate a variety of chromosome dynamics, including axial shortening, lateral compaction, and homolog pairing. However, how the in vivo activities of condensins are regulated and how functional interactors target condensins to chromatin are not well understood. To better understand how Drosophila melanogaster condensin is regulated, we performed a yeast two-hybrid screen and identified the chromo-barrel domain protein Mrg15 to interact with the Cap-H2 condensin subunit. Genetic interactions demonstrate that Mrg15 function is required for Cap-H2-mediated unpairing of polytene chromosomes in ovarian nurse cells and salivary gland cells. In diploid tissues, transvection assays demonstrate that Mrg15 inhibits transvection at Ubx and cooperates with Cap-H2 to antagonize transvection at yellow. In cultured cells, we show that levels of chromatin-bound Cap-H2 protein are partially dependent on Mrg15 and that Cap-H2-mediated homolog unpairing is suppressed by RNA interference depletion of Mrg15. Thus, maintenance of interphase chromosome compaction and homolog pairing status requires both Mrg15 and Cap-H2. We propose a model where the Mrg15 and Cap-H2 protein-protein interaction may serve to recruit Cap-H2 to chromatin and facilitates compaction of interphase chromatin.

SCFSlimb ubiquitin ligase suppresses condensin II-mediated nuclear reorganization by degrading Cap-H2.

Condensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCF(Slimb) ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCF(Slimb) function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCF(Slimb)-mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus.