Genetic and transcriptional evolution alters cancer cell line drug response.

Human cancer cell lines are the workhorse of cancer research. Although cell lines are known to evolve in culture, the extent of the resultant genetic and transcriptional heterogeneity and its functional consequences remain understudied. Here we use genomic analyses of 106 human cell lines grown in two laboratories to show extensive clonal diversity. Further comprehensive genomic characterization of 27 strains of the common breast cancer cell line MCF7 uncovered rapid genetic diversification. Similar results were obtained with multiple strains of 13 additional cell lines. Notably, genetic changes were associated with differential activation of gene expression programs and marked differences in cell morphology and proliferation. Barcoding experiments showed that cell line evolution occurs as a result of positive clonal selection that is highly sensitive to culture conditions. Analyses of single-cell-derived clones demonstrated that continuous instability quickly translates into heterogeneity of the cell line. When the 27 MCF7 strains were tested against 321 anti-cancer compounds, we uncovered considerably different drug responses: at least 75% of compounds that strongly inhibited some strains were completely inactive in others. This study documents the extent, origins and consequences of genetic variation within cell lines, and provides a framework for researchers to measure such variation in efforts to support maximally reproducible cancer research.

Glucagon-like peptide 2 for intestinal stem cell and Paneth cell repair during graft-versus-host disease in mice and humans.

Acute graft-versus-host disease (GVHD) is a life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). Although currently used GVHD treatment regimens target the donor immune system, we explored here an approach that aims at protecting and regenerating Paneth cells (PCs) and intestinal stem cells (ISCs). Glucagon-like-peptide-2 (GLP-2) is an enteroendocrine tissue hormone produced by intestinal L cells. We observed that acute GVHD reduced intestinal GLP-2 levels in mice and patients developing GVHD. Treatment with the GLP-2 agonist, teduglutide, reduced de novo acute GVHD and steroid-refractory GVHD, without compromising graft-versus-leukemia (GVL) effects in multiple mouse models. Mechanistically GLP-2 substitution promoted regeneration of PCs and ISCs, which enhanced production of antimicrobial peptides and caused microbiome changes. GLP-2 expanded intestinal organoids and reduced expression of apoptosis-related genes. Low numbers of L cells in intestinal biopsies and high serum levels of GLP-2 were associated with a higher incidence of nonrelapse mortality in patients undergoing allo-HCT. Our findings indicate that L cells are a target of GVHD and that GLP-2-based treatment of acute GVHD restores intestinal homeostasis via an increase of ISCs and PCs without impairing GVL effects. Teduglutide could become a novel combination partner for immunosuppressive GVHD therapy to be tested in clinical trials.

Intestinal microbiota domination under extreme selective pressures characterized by metagenomic read cloud sequencing and assembly.

BACKGROUND: Low diversity of the gut microbiome, often progressing to the point of intestinal domination by a single species, has been linked to poor outcomes in patients undergoing hematopoietic cell transplantation (HCT). Our ability to understand how certain organisms attain intestinal domination over others has been restricted in part by current metagenomic sequencing technologies that are typically unable to reconstruct complete genomes for individual organisms present within a sequenced microbial community. We recently developed a metagenomic read cloud sequencing and assembly approach that generates improved draft genomes for individual organisms compared to conventional short-read sequencing and assembly methods. Herein, we applied metagenomic read cloud sequencing to four stool samples collected longitudinally from an HCT patient preceding treatment and over the course of heavy antibiotic exposure. RESULTS: Characterization of microbiome composition by taxonomic classification of reads reveals that that upon antibiotic exposure, the subject's gut microbiome experienced a marked decrease in diversity and became dominated by Escherichia coli. While diversity is restored at the final time point, this occurs without recovery of the original species and strain-level composition. Draft genomes for individual organisms within each sample were generated using both read cloud and conventional assembly. Read clouds were found to improve the completeness and contiguity of genome assemblies compared to conventional assembly. Moreover, read clouds enabled the placement of antibiotic resistance genes present in multiple copies both within a single draft genome and across multiple organisms. The occurrence of resistance genes associates with the timing of antibiotics administered to the patient, and comparative genomic analysis of the various intestinal E. coli strains across time points as well as the bloodstream isolate showed that the subject's E. coli bloodstream infection likely originated from the intestine. The E. coli genome from the initial pre-transplant stool sample harbors 46 known antimicrobial resistance genes, while all other species from the pre-transplant sample each contain at most 5 genes, consistent with a model of heavy antibiotic exposure resulting in selective outgrowth of the highly antibiotic-resistant E. coli. CONCLUSION: This study demonstrates the application and utility of metagenomic read cloud sequencing and assembly to study the underlying strain-level genomic factors influencing gut microbiome dynamics under extreme selective pressures in the clinical context of HCT.

Reflections on a journey: a retrospective of the ISCB Student Council symposium series.

This article describes the motivation, origin and evolution of the student symposia series organised by the ISCB Student Council. The meeting series started thirteen years ago in Madrid and has spread to four continents. The article concludes with the highlights of the most recent edition of annual Student Council Symposium held in conjunction with the 25th Conference on Intelligent Systems for Molecular Biology and the 16th European Conference on Computational Biology, in Prague, in July 2017.

The TaxUMAP atlas: Efficient display of large clinical microbiome data reveals ecological competition in protection against bacteremia.

Longitudinal microbiome data provide valuable insight into disease states and clinical responses, but they are challenging to mine and view collectively. To address these limitations, we present TaxUMAP, a taxonomically informed visualization for displaying microbiome states in large clinical microbiome datasets. We used TaxUMAP to chart a microbiome atlas of 1,870 patients with cancer during therapy-induced perturbations. Bacterial density and diversity were positively associated, but the trend was reversed in liquid stool. Low-diversity states (dominations) remained stable after antibiotic treatment, and diverse communities had a broader range of antimicrobial resistance genes than dominations. When examining microbiome states associated with risk for bacteremia, TaxUMAP revealed that certain Klebsiella species were associated with lower risk for bacteremia localize in a region of the atlas that is depleted in high-risk enterobacteria. This indicated a competitive interaction that was validated experimentally. Thus, TaxUMAP can chart comprehensive longitudinal microbiome datasets, enabling insights into microbiome effects on human health.

Rare transmission of commensal and pathogenic bacteria in the gut microbiome of hospitalized adults.

Bacterial bloodstream infections are a major cause of morbidity and mortality among patients undergoing hematopoietic cell transplantation (HCT). Although previous research has demonstrated that pathogens may translocate from the gut microbiome into the bloodstream to cause infections, the mechanisms by which HCT patients acquire pathogens in their microbiome have not yet been described. Here, we use linked-read and short-read metagenomic sequencing to analyze 401 stool samples collected from 149 adults undergoing HCT and hospitalized in the same unit over three years, many of whom were roommates. We use metagenomic assembly and strain-specific comparison methods to search for high-identity bacterial strains, which may indicate transmission between the gut microbiomes of patients. Overall, the microbiomes of patients who share time and space in the hospital do not converge in taxonomic composition. However, we do observe six pairs of patients who harbor identical or nearly identical strains of the pathogen Enterococcus faecium, or the gut commensals Akkermansia muciniphila and Hungatella hathewayi. These shared strains may result from direct transmission between patients who shared a room and bathroom, acquisition from a common hospital source, or transmission from an unsampled intermediate. We also identify multiple patients with identical strains of species commonly found in commercial probiotics, including Lactobacillus rhamnosus and Streptococcus thermophilus. In summary, our findings indicate that sharing of identical pathogens between the gut microbiomes of multiple patients is a rare phenomenon. Furthermore, the observed potential transmission of commensal, immunomodulatory microbes suggests that exposure to other humans may contribute to microbiome reassembly post-HCT.

A compilation of fecal microbiome shotgun metagenomics from hematopoietic cell transplantation patients.

Hospitalized patients receiving hematopoietic cell transplants provide a unique opportunity to study the human gut microbiome. We previously compiled a large-scale longitudinal dataset of fecal microbiota and associated metadata, but we had limited that analysis to taxonomic composition of bacteria from 16S rRNA gene sequencing. Here we augment those data with shotgun metagenomics. The compilation amounts to a nested subset of 395 samples compiled from different studies at Memorial Sloan Kettering. Shotgun metagenomics describes the microbiome at the functional level, particularly in antimicrobial resistances and virulence factors. We provide accession numbers that link each sample to the paired-end sequencing files deposited in a public repository, which can be directly accessed by the online services of PATRIC to be analyzed without the users having to download or transfer the files. Then, we show how shotgun sequencing enables the assembly of genomes from metagenomic data. The new data, combined with the metadata published previously, enables new functional studies of the microbiomes of patients with cancer receiving bone marrow transplantation.

Microbiota dynamics in a randomized trial of gut decontamination during allogeneic hematopoietic cell transplantation.

BACKGROUNDGut decontamination (GD) can decrease the incidence and severity of acute graft-versus-host disease (aGVHD) in murine models of allogeneic hematopoietic cell transplantation (HCT). In this pilot study, we examined the impact of GD on gut microbiome composition and the incidence of aGVHD in HCT patients.METHODSWe randomized 20 patients undergoing allogeneic HCT to receive (GD) or not receive (no-GD) oral vancomycin-polymyxin B from day -5 through neutrophil engraftment. We evaluated shotgun metagenomic sequencing of serial stool samples to compare the composition and diversity of the gut microbiome between study arms. We assessed clinical outcomes in the 2 arms and performed strain-specific analyses of pathogens that caused bloodstream infections (BSI).RESULTSThe 2 arms did not differ in the predefined primary outcome of Shannon diversity of the gut microbiome at 2 weeks post-HCT (genus, P = 0.8; species, P = 0.44) or aGVHD incidence (P = 0.58). Immune reconstitution of T cell and B cell subsets was similar between groups. Five patients in the no-GD arm had 8 BSI episodes versus 1 episode in the GD arm (P = 0.09). The BSI-causing pathogens were traceable to the gut in 7 of 8 BSI episodes in the no-GD arm, including Staphylococcus species.CONCLUSIONWhile GD did not differentially affect Shannon diversity or clinical outcomes, our findings suggest that GD may protect against gut-derived BSI in HCT patients by decreasing the prevalence or abundance of gut pathogens.TRIAL REGISTRATIONClinicalTrials.gov NCT02641236.FUNDINGNIH, Damon Runyon Cancer Research Foundation, V Foundation, Sloan Foundation, Emerson Collective, and Stanford Maternal & Child Health Research Institute.

Human ß-defensin 2 ameliorates acute GVHD by limiting ileal neutrophil infiltration and restraining T cell receptor signaling.

Acute graft-versus-host disease (aGVHD), which is driven by allogeneic T cells, has a high mortality rate and limited treatment options. Human ß-defensin 2 (hBD-2) is an endogenous epithelial cell-derived host-defense peptide. In addition to its antimicrobial effects, hBD-2 has immunomodulatory functions thought to be mediated by CCR2 and CCR6 in myeloid cells. In this study, we analyzed the effect of recombinant hBD-2 on aGVHD development. We found that intestinal ß-defensin expression was inadequately induced in response to inflammation in two independent cohorts of patients with aGVHD and in a murine aGVHD model. Treatment of mice with hBD-2 reduced GVHD severity and mortality and modulated the intestinal microbiota composition, resulting in reduced neutrophil infiltration in the ileum. Furthermore, hBD-2 treatment decreased proliferation and proinflammatory cytokine production by allogeneic T cells in vivo while preserving the beneficial graft-versus-leukemia effect. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation, and metabolism in a CCR2- and CCR6-independent manner by reducing proximal T cell receptor signaling. Furthermore, hBD-2 treatment diminished alloreactive T cell infiltration and the expression of genes involved in T cell receptor signaling in the ilea of mice with aGVHD. Together, we found that both human and murine aGVHD were characterized by a lack of intestinal ß-defensin induction and that recombinant hBD-2 represents a potential therapeutic strategy to counterbalance endogenous hBD-2 deficiency.

Acquisition, transmission and strain diversity of human gut-colonizing crAss-like phages.

CrAss-like phages are double-stranded DNA viruses that are prevalent in human gut microbiomes. Here, we analyze gut metagenomic data from mother-infant pairs and patients undergoing fecal microbiota transplantation to evaluate the patterns of acquisition, transmission and strain diversity of crAss-like phages. We find that crAss-like phages are rarely detected at birth but are increasingly prevalent in the infant microbiome after one month of life. We observe nearly identical genomes in 50% of cases where the same crAss-like clade is detected in both the mother and the infant, suggesting vertical transmission. In cases of putative transmission of prototypical crAssphage (p-crAssphage), we find that a subset of strains present in the mother are detected in the infant, and that strain diversity in infants increases with time. Putative tail fiber proteins are enriched for nonsynonymous strain variation compared to other genes, suggesting a potential evolutionary benefit to maintaining strain diversity in specific genes. Finally, we show that p-crAssphage can be acquired through fecal microbiota transplantation.

Author Correction: Acquisition, transmission and strain diversity of human gut-colonizing crAss-like phages.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Bioinformatic Analysis of Integrative Mobile Genetic Elements Highlights Their Role in Bacterial Adaptation.

Mobile genetic elements (MGEs) contribute to bacterial adaptation and evolution; however, high-throughput, unbiased MGE detection remains challenging. We describe MGEfinder, a bioinformatic toolbox that identifies integrative MGEs and their insertion sites by using short-read sequencing data. MGEfinder identifies the genomic site of each MGE insertion and infers the identity of the inserted sequence. We apply MGEfinder to 12,374 sequenced isolates of 9 prevalent bacterial pathogens, including Mycobacterium tuberculosis, Staphylococcus aureus, and Escherichia coli, and identify thousands of MGEs, including candidate insertion sequences, conjugative transposons, and prophage elements. The MGE repertoire and insertion rates vary across species, and integration sites often cluster near genes related to antibiotic resistance, virulence, and pathogenicity. MGE insertions likely contribute to antibiotic resistance in laboratory experiments and clinical isolates. Additionally, we identified thousands of mobility genes, a subset of which have unknown function opening avenues for exploration. Future application of MGEfinder to commensal bacteria will further illuminate bacterial adaptation and evolution.

Whole-blood dysregulation of actin-cytoskeleton pathway in adult spinal muscular atrophy patients.

OBJECTIVE: Recent advances in therapeutics have improved prognosis for severely affected spinal muscular atrophy (SMA) type 1 and 2 patients, while the best method of treatment for SMA type 3 patients with later onset of disease is unknown. To better characterize the SMA type 3 population and provide potential therapeutic targets, we aimed to understand gene expression differences in whole blood of SMA type 3 patients (n = 31) and age- and gender-matched controls (n = 34). METHODS: We performed the first large-scale whole blood transcriptomic screen with L1000, a rapid, high-throughput gene expression profiling technology that uses 978 landmark genes to capture a representation of the transcriptome and predict expression of 9196 additional genes. RESULTS: The primary downregulated KEGG pathway in adult SMA type 3 patients was "Regulation of Actin Cytoskeleton," and downregulated expression of key genes in this pathway, including ROCK1, RHOA, and ACTB, was confirmed in the same whole blood samples using RT-qPCR. SMA type 3 patient-derived fibroblasts had lower expression of these genes compared to control fibroblasts from unaffected first-degree relatives. Overexpression of SMN levels using an AAV vector in fibroblasts did not normalize ROCK1, RHOA, and ACTB mRNA expression, indicating the involvement of additional genes in cytoskeleton dynamic regulation. INTERPRETATION: Our findings from whole blood and patient-derived fibroblasts suggest SMA type 3 patients have decreased expression of actin cytoskeleton regulators. These observations provide new insights and potential therapeutic targets for SMA patients with longstanding denervation and secondary musculoskeletal pathophysiology.

Read cloud sequencing elucidates microbiome dynamics in a hematopoietic cell transplant patient.

Low intestinal microbial diversity, often leading to domination of the intestine by a single organism, is associated with poor outcomes following hematopoietic cell transplantation (HCT). Understanding how certain organisms achieve domination in the intestine is limited by current metagenomic sequencing technologies, which are typically unable to reconstruct complete genome drafts without bacterial isolation and culture. Recently, we developed a metagenomic read cloud sequencing approach that provides significantly improved genome drafts for individual organisms compared to conventional short-read sequencing methods. Here, we apply read cloud sequencing to four longitudinal stool samples collected from an HCT patient before and after heavy antibiotic exposure. During this time period, the patient experienced Escherichia coli gut domination and an E. coli bloodstream infection. We find that read clouds enable the placement of multiple copies of antibiotic resistance genes both within and across genomes, and the presence of resistance genes correlates with the timing of antibiotics administered to the patient. Comparative genomic analysis reveals that the E. coli bloodstream infection likely originated from the gut. The pre-transplant E. coli genome harbors 46 known resistance genes, whereas all other organisms from the pre-transplant time point contain 5 or fewer resistance genes, supporting a model in which the E. coli outgrowth was a result of selection by heavy antibiotic exposure. This case study highlights the application of metagenomic read cloud sequencing in a clinical context to elucidate the genomic underpinnings of microbiome dynamics under extreme selective pressures.

Reorganization of chromosome architecture in replicative cellular senescence.

Replicative cellular senescence is a fundamental biological process characterized by an irreversible arrest of proliferation. Senescent cells accumulate a variety of epigenetic changes, but the three-dimensional (3D) organization of their chromatin is not known. We applied a combination of whole-genome chromosome conformation capture (Hi-C), fluorescence in situ hybridization, and in silico modeling methods to characterize the 3D architecture of interphase chromosomes in proliferating, quiescent, and senescent cells. Although the overall organization of the chromatin into active (A) and repressive (B) compartments and topologically associated domains (TADs) is conserved between the three conditions, a subset of TADs switches between compartments. On a global level, the Hi-C interaction matrices of senescent cells are characterized by a relative loss of long-range and gain of short-range interactions within chromosomes. Direct measurements of distances between genetic loci, chromosome volumes, and chromatin accessibility suggest that the Hi-C interaction changes are caused by a significant reduction of the volumes occupied by individual chromosome arms. In contrast, centromeres oppose this overall compaction trend and increase in volume. The structural model arising from our study provides a unique high-resolution view of the complex chromosomal architecture in senescent cells.

Tetranucleotide usage highlights genomic heterogeneity among mycobacteriophages.

Background The genomic sequences of mycobacteriophages, phages infecting mycobacterial hosts, are diverse and mosaic. Mycobacteriophages often share little nucleotide similarity, but most of them have been grouped into lettered clusters and further into subclusters. Traditionally, mycobacteriophage genomes are analyzed based on sequence alignment or knowledge of gene content. However, these approaches are computationally expensive and can be ineffective for significantly diverged sequences. As an alternative to alignment-based genome analysis, we evaluated tetranucleotide usage in mycobacteriophage genomes. These methods make it easier to characterize features of the mycobacteriophage population at many scales. Description We computed tetranucleotide usage deviation (TUD), the ratio of observed counts of 4-mers in a genome to the expected count under a null model. TUD values are comparable between members of a phage subcluster and distinct between subclusters. With few exceptions, neighbor joining phylogenetic trees and hierarchical clustering dendrograms constructed using TUD values place phages in a monophyletic clade with members of the same subcluster. Regions in a genome with exceptional TUD values can point to interesting features of genomic architecture. Finally, we found that subcluster B3 mycobacteriophages contain significantly overrepresented 4-mers and 6-mers that are atypical of phage genomes. Conclusions Statistics based on tetranucleotide usage support established clustering of mycobacteriophages and can uncover interesting relationships within and between sequenced phage genomes. These methods are efficient to compute and do not require sequence alignment or knowledge of gene content. The code to download mycobacteriophage genome sequences and reproduce our analysis is freely available at https://github.com/bsiranosian/tango_final.