Comprehensive statistical inference of the clonal structure of cancer from multiple biopsies.

A comprehensive characterization of tumor genetic heterogeneity is critical for understanding how cancers evolve and escape treatment. Although many algorithms have been developed for capturing tumor heterogeneity, they are designed for analyzing either a single type of genomic aberration or individual biopsies. Here we present THEMIS (Tumor Heterogeneity Extensible Modeling via an Integrative System), which allows for the joint analysis of different types of genomic aberrations from multiple biopsies taken from the same patient, using a dynamic graphical model. Simulation experiments demonstrate higher accuracy of THEMIS over its ancestor, TITAN. The heterogeneity analysis results from THEMIS are validated with single cell DNA sequencing from a clinical tumor biopsy. When THEMIS is used to analyze tumor heterogeneity among multiple biopsies from the same patient, it helps to reveal the mutation accumulation history, track cancer progression, and identify the mutations related to treatment resistance. We implement our model via an extensible modeling platform, which makes our approach open, reproducible, and easy for others to extend.

A Distributed Network for Intensive Longitudinal Monitoring in Metastatic Triple-Negative Breast Cancer.

Accelerating cancer research is expected to require new types of clinical trials. This report describes the Intensive Trial of OMics in Cancer (ITOMIC) and a participant with triple-negative breast cancer metastatic to bone, who had markedly elevated circulating tumor cells (CTCs) that were monitored 48 times over 9 months. A total of 32 researchers from 14 institutions were engaged in the patient's evaluation; 20 researchers had no prior involvement in patient care and 18 were recruited specifically for this patient. Whole-exome sequencing of 3 bone marrow samples demonstrated a novel ROS1 variant that was estimated to be present in most or all tumor cells. After an initial response to cisplatin, a hypothesis of crizotinib sensitivity was disproven. Leukapheresis followed by partial CTC enrichment allowed for the development of a differential high-throughput drug screen and demonstrated sensitivity to investigational BH3-mimetic inhibitors of BCL-2 that could not be tested in the patient because requests to the pharmaceutical sponsors were denied. The number and size of CTC clusters correlated with clinical status and eventually death. Focusing the expertise of a distributed network of investigators on an intensively monitored patient with cancer can generate high-resolution views of the natural history of cancer and suggest new opportunities for therapy. Optimization requires access to investigational drugs.

A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo.

RNAs must assemble into specific structures in order to carry out their biological functions, but in vitro RNA folding reactions produce multiple misfolded structures that fail to exchange with functional structures on biological time scales. We used carefully designed self-cleaving mRNAs that assemble through well-defined folding pathways to identify factors that differentiate intracellular and in vitro folding reactions. Our previous work showed that simple base-paired RNA helices form and dissociate with the same rate and equilibrium constants in vivo and in vitro. However, exchange between adjacent secondary structures occurs much faster in vivo, enabling RNAs to quickly adopt structures with the lowest free energy. We have now used this approach to probe the effects of an extensively characterized DEAD-box RNA helicase, Mss116p, on a series of well-defined RNA folding steps in yeast. Mss116p overexpression had no detectable effect on helix formation or dissociation kinetics or on the stability of interdomain tertiary interactions, consistent with previous evidence that intracellular factors do not affect these folding parameters. However, Mss116p overexpression did accelerate exchange between adjacent helices. The nonprocessive nature of RNA duplex unwinding by DEAD-box RNA helicases is consistent with a branch migration mechanism in which Mss116p lowers barriers to exchange between otherwise stable helices by the melting and annealing of one or two base pairs at interhelical junctions. These results suggest that the helicase activity of DEAD-box proteins like Mss116p distinguish intracellular RNA folding pathways from nonproductive RNA folding reactions in vitro and allow RNA structures to overcome kinetic barriers to thermodynamic equilibration in vivo.

NLRP7 affects trophoblast lineage differentiation, binds to overexpressed YY1 and alters CpG methylation.

Maternal-effect mutations in NLRP7 cause rare biparentally inherited hydatidiform moles (BiHMs), abnormal pregnancies containing hypertrophic vesicular trophoblast but no embryo. BiHM trophoblasts display abnormal DNA methylation patterns affecting maternally methylated germline differentially methylated regions (gDMRs), suggesting that NLRP7 plays an important role in reprogramming imprinted gDMRs. How NLRP7-a component of the CATERPILLAR family of proteins involved in innate immunity and apoptosis-causes these specific DNA methylation and trophoblast defects is unknown. Because rodents lack NLRP7, we used human embryonic stem cells to study its function and demonstrate that NLRP7 interacts with YY1, an important chromatin-binding factor. Reduced NLRP7 levels alter DNA methylation and accelerate trophoblast lineage differentiation. NLRP7 thus appears to function in chromatin reprogramming and DNA methylation in the germline or early embryonic development, functions not previously associated with members of the NLRP family.

mRNA secondary structures fold sequentially but exchange rapidly in vivo.

RNAs adopt defined structures to perform biological activities, and conformational transitions among alternative structures are critical to virtually all RNA-mediated processes ranging from metabolite-activation of bacterial riboswitches to pre-mRNA splicing and viral replication in eukaryotes. Mechanistic analysis of an RNA folding reaction in a biological context is challenging because many steps usually intervene between assembly of a functional RNA structure and execution of a biological function. We developed a system to probe mechanisms of secondary structure folding and exchange directly in vivo using self-cleavage to monitor competition between mutually exclusive structures that promote or inhibit ribozyme assembly. In previous work, upstream structures were more effective than downstream structures in blocking ribozyme assembly during transcription in vitro, consistent with a sequential folding mechanism. However, upstream and downstream structures blocked ribozyme assembly equally well in vivo, suggesting that intracellular folding outcomes reflect thermodynamic equilibration or that annealing of contiguous sequences is favored kinetically. We have extended these studies to learn when, if ever, thermodynamic stability becomes an impediment to rapid equilibration among alternative RNA structures in vivo. We find that a narrow thermodynamic threshold determines whether kinetics or thermodynamics govern RNA folding outcomes in vivo. mRNA secondary structures fold sequentially in vivo, but exchange between adjacent secondary structures is much faster in vivo than it is in vitro. Previous work showed that simple base-paired RNA helices dissociate at similar rates in vivo and in vitro so exchange between adjacent structures must occur through a different mechanism, one that likely involves facilitation of branch migration by proteins associated with nascent transcripts.

Proteomic identification of tmRNA substrates.

The tmRNA-SmpB system releases ribosomes stalled on truncated mRNAs and tags the nascent polypeptides to target them for proteolysis. In many species, mutations that disrupt tmRNA activity cause defects in growth or development. In Caulobacter crescentus cells lacking tmRNA activity there is a delay in the initiation of DNA replication, which disrupts the cell cycle. To understand the molecular basis for this phenotype, 73 C. crescentus proteins were identified that are tagged by tmRNA under normal growth conditions. Among these substrates, proteins involved in DNA replication, recombination, and repair were overrepresented, suggesting that misregulation of these factors in the absence of tmRNA activity might be responsible for the delay in initiation of DNA replication. Analysis of the tagging sites within these substrates revealed a conserved nucleotide motif 5' of the tagging site, which is required for wild-type tmRNA tagging.

Kinetics and thermodynamics make different contributions to RNA folding in vitro and in yeast.

RNAs somehow adopt specific functional structures despite the capacity to form alternative nonfunctional structures with similar stabilities. We analyzed RNA assembly during transcription in vitro and in yeast using hairpin ribozyme self-cleavage to assess partitioning between functional ribozyme structures and nonfunctional stem loops. Complementary insertions located upstream of the ribozyme inhibited ribozyme assembly more than downstream insertions during transcription in vitro, consistent with a sequential folding model in which the outcome is determined by the structure that forms first. In contrast, both upstream and downstream insertions strongly inhibited assembly of the same ribozyme variants when expressed as chimeric mRNAs in yeast, indicating that inhibitory stem loops can form even after the entire ribozyme sequence has been transcribed. Evidently, some feature unique to the intracellular environment modulates the influence of transcription polarity and enhances the contribution of thermodynamic stability to RNA folding in vivo.

Kinetic analysis of ribozyme-substrate complex formation in yeast.

Many RNA-mediated reactions in transcription, translation, RNA processing, and transport require assembly of RNA complexes, yet assembly pathways remain poorly understood. Assembly mechanisms can be difficult to assess in a biological context because many components interact in complex pathways and individual steps are difficult to isolate experimentally. Our previous studies of self-cleaving hairpin ribozymes showed that kinetic and equilibrium parameters measured in yeast agree well with parameters measured in vitro under ionic conditions that mimic the intracellular environment. We now report studies of intermolecular reactions with ribozyme and target sequences expressed in yeast as separate chimeric U3 snoRNAs. In this system, intracellular cleavage rates reflect the kinetics of ribozyme-substrate complex formation through annealing of base-paired helices. Second-order rate constants increased with increasing helix length for in vitro reactions with 2 mM MgCl(2) and 150 mM NaCl and in vivo but not in reactions with 10 mM MgCl(2). Thus, efficient RNA complex formation required a larger extent of complementarity in vivo than in vitro under conditions with high concentrations of divalent cations. The most efficient intracellular cleavage reactions exhibited second-order rate constants that were 15- to 30-fold below rate constants for cleavage of oligonucleotides in vitro. Careful analysis of structural features that influence cleavage efficiency points to substrate binding as the rate-determining step in the intracellular cleavage pathway. Second-order rate constants for intermolecular cleavage agree well with diffusion coefficients reported for U3 snoRNPs in vivo suggesting that complex formation between chimeric ribozyme and substrate snoRNPs in yeast nuclei is diffusion limited.