Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq.

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3' ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3' ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

Osteosarcoma: Novel prognostic biomarkers using circulating and cell-free tumour DNA.

AIM: Osteosarcoma (OS) is the most common primary bone tumour in children and adolescents. Circulating free (cfDNA) and circulating tumour DNA (ctDNA) are promising biomarkers for disease surveillance and prognostication in several cancer types; however, few such studies are reported for OS. The purpose of this study was to discover and validate methylation-based biomarkers to detect plasma ctDNA in patients with OS and explore their utility as prognostic markers. METHODS: Candidate CpG markers were selected through analysis of methylation array data for OS, non-OS tumours and germline samples. Candidates were validated in two independent OS datasets (n = 162, n = 107) and the four top-performing markers were selected. Methylation-specific digital droplet PCR (ddPCR) assays were designed and experimentally validated in OS tumour samples (n = 20) and control plasma samples. Finally, ddPCR assays were applied to pre-operative plasma and where available post-operative plasma from 72 patients with OS, and findings correlated with outcome. RESULTS: Custom ddPCR assays detected ctDNA in 69% and 40% of pre-operative plasma samples (n = 72), based on thresholds of one or two positive markers respectively. ctDNA was detected in 5/17 (29%) post-operative plasma samples from patients, which in four cases were associated with or preceded disease relapse. Both pre-operative cfDNA levels and ctDNA detection independently correlated with overall survival (p = 0.0015 and p = 0.0096, respectively). CONCLUSION: Our findings illustrate the potential of mutation-independent methylation-based ctDNA assays for OS. This study lays the foundation for multi-institutional collaborative studies to explore the utility of plasma-derived biomarkers in the management of OS.

RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition.

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.

Development of a cell-free split-luciferase biochemical assay as a tool for screening for inhibitors of challenging protein-protein interaction targets.

Targeting the interaction of proteins with weak binding affinities or low solubility represents a particular challenge for drug screening. The NanoLuc â ® Binary Technology (NanoBiT â ®) was originally developed to detect protein-protein interactions in live mammalian cells. Here we report the successful translation of the NanoBit cellular assay into a biochemical, cell-free format using mammalian cell lysates. We show that the assay is suitable for the detection of both strong and weak protein interactions such as those involving the binding of RAS oncoproteins to either RAF or phosphoinositide 3-kinase (PI3K) effectors respectively, and that it is also effective for the study of poorly soluble protein domains such as the RAS binding domain of PI3K. Furthermore, the RAS interaction assay is sensitive and responds to both strong and weak RAS inhibitors. Our data show that the assay is robust, reproducible, cost-effective, and can be adapted for small and large-scale screening approaches. The NanoBit Biochemical Assay offers an attractive tool for drug screening against challenging protein-protein interaction targets, including the interaction of RAS with PI3K.