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Crowd-sourced benchmarking of single-sample tumor subclonal reconstruction.
No full textSubclonal reconstruction algorithms use bulk DNA sequencing data to quantify parameters of tumor evolution, allowing an assessment of how cancers initiate, progress and respond to selective pressures. We launched the ICGC-TCGA (International Cancer Genome Consortium-The Cancer Genome Atlas) DREAM Somatic Mutation Calling Tumor Heterogeneity and Evolution Challenge to benchmark existing subclonal reconstruction algorithms. This 7-year community effort used cloud computing to benchmark 31 subclonal reconstruction algorithms on 51 simulated tumors. Algorithms were scored on seven independent tasks, leading to 12,061 total runs. Algorithm choice influenced performance substantially more than tumor features but purity-adjusted read depth, copy-number state and read mappability were associated with the performance of most algorithms on most tasks. No single algorithm was a top performer for all seven tasks and existing ensemble strategies were unable to outperform the best individual methods, highlighting a key research need. All containerized methods, evaluation code and datasets are available to support further assessment of the determinants of subclonal reconstruction accuracy and development of improved methods to understand tumor evolution
Population pharmacokinetics of rifampicin in plasma and cerebrospinal fluid in adults with tuberculosis meningitis.
No full textBACKGROUND: Several ongoing clinical trials are evaluating high-dose rifampicin (up to 35 mg/kg) for tuberculous meningitis (TBM). However, rifampicin pharmacokinetics at higher doses is not fully characterized, particularly in cerebrospinal fluid (CSF), the site of TBM disease. METHODS: In a randomized controlled trial, adults with HIV-associated TBM were assigned to experimental arms of high-dose rifampicin (oral, 35 mg/kg; intravenous, 20 mg/kg) plus linezolid, with or without aspirin, or a control arm that received the standard of care with 10 mg/kg of oral rifampicin. Rifampicin concentrations, including the unbound fraction, were measured on plasma samples, and CSF was collected on days 3 and 28 of study enrollment. Data were analyzed by nonlinear mixed effects modeling. RESULTS: In total, 400 plasma and 44 CSF rifampicin concentrations from 48 participants were used for model development. The median (range) age and weight were 39 years (25-78) and 60 kg (30-107). Rifampicin pharmacokinetics was best described by a 2-compartment disposition model with first-order transit oral absorption and elimination via saturable hepatic extraction. Typical clearance values for the standard dose for days 3 and 28 were 33.1 and 41.4 L/h, respectively; high-dose values were 46.1 and 70.2 L/h. The CSF-plasma ratio was approximately 6% and the equilibration half-life was 3.2 hours. Simulated standard-dose rifampicin did not reach CSF concentrations above the critical concentration for Mycobacterium tuberculosis. CONCLUSIONS: CSF penetration with standard-dose rifampicin is low. Our findings support continued evaluation of high-dose rifampicin for TBM treatment
Replisome passage through the cohesin ring
No full textFollowing eukaryotic genome replication, the ring-shaped cohesin complex embraces the two newly synthesized sister chromatids, enabling their faithful segregation during cell divisions. Replisome passage through cohesin rings has been envisioned as a fail-safe mechanism that ensures co-entrapment of replication products—whether replisomes can indeed pass through cohesin rings remains unknown. Here, we use biochemical reconstitution and single-molecule fluorescence microscopy to directly visualize replisome-cohesin encounters. We find that the translocating eukaryotic replicative Cdc45-Mcm2-7-GINS (CMG) helicase, unlike other obstacles of similar size, readily passes through cohesin rings. Fully reconstituted replisomes also pass cohesin rings to leave both replication products trapped inside. Replisome passage is primarily aided by DNA polymerases α and ε, a finding that necessitates re-evaluation of canonical cohesion establishment factor roles. Our findings demonstrate the existence of a simple mechanismthat links genome replication with chromosome segregation: replisome passage through cohesin rings
TCF1 and LEF1 promote B-1a cell homeostasis and regulatory function.
No full textB-1 cells are innate-like immune cells abundant in serosal cavities with antibodies enriched in bacterial recognition, yet their existence in humans has been controversial1-3. The CD5+ B-1a subset expresses anti-inflammatory molecules including IL-10, PDL1 and CTLA4 and can be immunoregulatory4-6. Unlike conventional B cells that are continuously replenished, B-1a cells are produced early in life and maintained through self-renewal7. Here we show that the transcription factors TCF1 and LEF1 are critical regulators of B-1a cells. LEF1 expression is highest in fetal and bone marrow B-1 progenitors, whereas the levels of TCF1 are higher in splenic and peritoneal B-1 cells than in B-1 progenitors. TCF1-LEF1 double deficient mice have reduced B-1a cells and defective B-1a cell maintenance. These transcription factors promote MYC-dependent metabolic pathways and induce a stem-like population upon activation, partly via IL-10 production. In the absence of TCF1 and LEF1, B-1 cells proliferate excessively and acquire an exhausted phenotype with reduced IL-10 and PDL1 expression. Furthermore, adoptive transfer of B-1 cells lacking TCF1 and LEF1 fails to suppress brain inflammation. These transcription factors are also expressed in human chronic lymphocytic leukaemia B cells and in a B-1-like population that is abundant in pleural fluid and circulation of some patients with pleural infection. Our findings define a TCF1-LEF1-driven transcriptional program that integrates stemness and regulatory function in B-1a cells
Evolution of a melanoma that escapes allogeneic rejection.
No full textThe histocompatibility barrier prevents the transfer of both normal and tumor cells between individuals; however, clonally transmissible cancers in dogs, Tasmanian devils, and soft-shell clams can naturally transmit as allografts. To understand if cancer cells can more generally evolve to escape the histocompatibility barrier, we have serially passaged a mouse melanoma into increasingly mismatched mouse strains until a transplantable tumor emerged. The transplantable melanoma cells are characterized by an antiviral immune signature and the upregulation of endogenous retrotransposable elements (RTEs), major histocompatibility complex class I (MHC class I), programmed cell death ligand-1 (PD-L1), and Qa-1 non-classical MHC molecules. Knockout of the RNA sensor retinoic acid-inducible gene I (RIG-I) reduces expression of PD-L1 and Qa-1, and antibody-mediated blockade of PD-L1 and Qa-1 induces tumor rejection. Thus, an immune antiviral signature linked to RTEs upregulation facilitates escape of the melanoma from allogeneic rejection, simultaneously making the tumor sensitive to PD-L1 and Qa-1 antagonism. A similar immune signature is found in human melanomas that respond to PD-L1 blockade
Predictors and outcomes of non-small cell lung carcinoma patients following severe immune checkpoint inhibitor toxicity: A real-world UK multi-centre study.
No full textPURPOSE: Evaluation of predictors and outcomes in NSCLC patients treated with an immune checkpoint inhibitor (ICI) following a severe immune-related adverse event (irAE). METHODS: We included all NSCLC patients receiving ≥1 ICI cycle and corticosteroids for CTCAE Grade ≥3 irAEs between 2017 and 2023 from three UK NHS teaching hospitals. Progression-free survival (PFS) and overall survival (OS) after the 1st irAE, best overall response (BOR) to ICI, and predictors of clinical benefit were evaluated. Kaplan-Meier, Cox and logistic regression models, and Wilcoxon tests were used. RESULTS: We screened 1658 NSCLC patients and identified 80 eligible subjects. The majority of patients had metastatic (n = 50, 63%) vs. localized (n = 30, 37%) NSCLC. Most patients developed a single ≥Grade 3 irAE on 1st line ICI (n = 71, 89%). Overall, 14 (18%) patients developed 2nd irAEs, 7 after rechallenge with ICIs. In the complete cohort, median OS after 1st irAE was 15.84 months (95% CI, 12.45-26.91). Lower neutrophil-to-lymphocyte ratio (NLR), patients receiving >4 cycles of ICI or median duration of ICI of >2.76 months before 1st irAE were associated with improved OS (p < 0.05), the latter two with PFS (p < 0.05). Age, gender, stage, KRAS mutation, PD-L1 and ICI type were not associated with PFS or OS. Pneumonitis as 1st irAE had the worst PFS and OS (p < 0.05). Median starting corticosteroid dose of ≤60 mg for 1st irAE had an improved OS (p = 0.04). Post 1st irAE response associated with better PFS and OS (p < 0.05). Number and duration of irAEs and additional immunosuppressive agents (14% of patients) were not associated with PFS or OS. CONCLUSIONS: In ≥Grade 3 irAEs patients managed with corticosteroids, lower baseline NLR, longer ICI use, response to ICI after 1st irAE, and a ≤60 mg corticosteroid dose had promising outcomes
Cell cycle regulation has shaped replication origins in budding yeast.
No full textEukaryotic DNA replication initiates from genomic loci known as origins. At budding yeast origins like ARS1, a double hexamer (DH) of the MCM replicative helicase is assembled by origin recognition complex (ORC), Cdc6 and Cdt1 by sequential hexamer loading from two opposed ORC binding sites. Cyclin-dependent kinase (CDK) inhibits DH assembly, which prevents re-replication by restricting helicase loading to the G1 phase. Here, we show that an intrinsically disordered region (IDR) in the Orc2 subunit promotes interaction between ORC and the first loaded, closed-ring MCM hexamer (the MCM-ORC (MO) intermediate). CDK-dependent phosphorylation of this IDR blocks MO formation and DH assembly. We show that MO stabilizes ORC at lower-affinity binding sites required for second hexamer loading. Origins comprising two high-affinity ORC sites can assemble DH efficiently without MO by independently loading single hexamers. Strikingly, these origins escape CDK inhibition in vitro and in vivo. Our work reveals mechanistic plasticity in MCM loading with implications for understanding how CDK regulation has shaped yeast origin evolution and how natural, strong origins might escape cell cycle regulation. We also identify key steps common to loading pathways, with implications for understanding how MCM is loaded in other eukaryotes
A human induced pluripotent stem cell toolbox for studying sex chromosome effects.
No full textSex chromosomes shape male (XY)-female (XX) differences in development and disease. These differences can be modeled in vitro by comparing XY and XX human induced pluripotent stem cells (hiPSCs). However, in this system, inter-individual autosomal variation and unstable X-dosage compensation can confound identification of sex chromosomal effects. Here, we utilize sex chromosome loss in XXY fibroblasts to generate XX and XY hiPSCs that are autosomally isogenic and exhibit stable X-dosage compensation. We also create X-monosomic (XO) hiPSCs, to investigate X-Y dosage effects. Using these autosomally isogenic lines, we examine sex differences in pluripotent stem cell expression. Transcriptional differences between XX and XY hiPSCs are surprisingly modest. However, X-haploinsufficiency induces transcriptional deregulation predominantly affecting autosomes. This effect is mediated by Y-genes with broad housekeeping functions that have X-homologs escaping X inactivation. Our isogenic hiPSC lines provide a resource for exploring sex chromosome effects on development and disease in vitro
Nonredundant roles of topoisomerase 2α and 2β in the cytosolic replication of vaccinia virus.
No full textVaccinia virus is a large enveloped DNA virus, which, like all poxviruses, replicates in the cytoplasm of infected cells. Vaccinia was historically thought to encode all the proteins required for its replication. However, more recent findings have shown that nuclear host proteins are redirected to the cytoplasm to facilitate viral replication. Among these, topoisomerase 2α (TOP2A) and 2β (TOP2B), which mediate nuclear transcription, DNA replication, and chromosome segregation are the most abundant host proteins associated with nascent viral genomes. Here, we investigate the mechanisms driving TOP2A and TOP2B cytoplasmic translocation and their role in viral replication. We found that early viral protein synthesis induces the cytosolic relocalization of both isoforms, which are subsequently recruited to viral factories by an interaction of their C-terminal domains with the viral ligase, A50. TOP2A promotes replication by interacting with the vaccinia DNA replication machinery. In contrast, TOP2B suppresses replication by enhancing the formation of double-stranded RNA and antiviral granules, containing components of the tRNA splicing ligase complex. Our analysis provides new insights into host-pathogen interactions during poxvirus infection and the role of topoisomerase 2 outside of the nucleus
ESCRT-III function in membrane fission and repair.
No full textThe endosomal sorting complex required for transport (ESCRT) machinery is an evolutionarily conserved multisubunit protein complex that remodels cellular membranes. Beyond its classical role in endosomal sorting, the ESCRT machinery has been implicated in an ever-growing number of functions, including viral budding, cytokinesis, autophagy, extracellular vesicle release, pruning of synaptic processes and the repair and closure of holes in cellular membranes. Membrane remodelling functions are typically ascribed to the ESCRT-III subcomplex. In this Review, we discuss recent mechanistic and structural insights into how these proteins assemble and are remodelled to achieve membrane severing. We focus particularly on how ESCRT-III is engaged at different subcellular compartments during both interphase and mitosis to repair and remodel membranes