Summary The Mre11/Rad50/NBS1 (MRN) complex plays many roles in response to DNA double strand breaks (DSBs), but its functions in repair by non homologous end joining (NHEJ) pathways are poorly understood. We have investigated requirements for MRN in Class Switch Recombination (CSR), a programmed DNA rearrangement in B lymphocytes that requires NHEJ. To this end we have engineered mice that lack the entire MRN complex in B lymphocytes, or possess an intact complex harboring mutant Mre11 lacking DNA nuclease activities. MRN deficiency confers a striking defect in CSR, impacting both the Classic and Alternative NHEJ pathways. In contrast, absence of Mre11 nuclease activities causes a milder phenotype, revealing a separation of function within the complex. We propose a model in which MRN stabilizes distant breaks and processes DNA termini to facilitate repair by both the Classical and Alternative NHEJ pathways.

Hypomorphic mutations in the genes encoding the MRE11/RAD50/NBS1 (MRN) DNA repair complex lead to cancer-prone syndromes. MRN binds DNA double strand breaks where it functions in repair and triggers cell cycle checkpoints via activation of the ataxia-telangiectasia mutated (ATM) kinase. To gain understanding of MRN in cancer, we engineered mice with B lymphocytes lacking MRN, or harboring MRN in which MRE11 lacks nuclease activities. Both forms of MRN deficiency led to hallmarks of cancer, including oncogenic translocations involving c-Myc and the immunoglobulin locus. These pre-neoplastic B lymphocytes did not progress to detectable B lineage lymphoma, even in the absence of p53. Moreover, Mre11 deficiencies prevented tumorigenesis in a mouse model strongly predisposed to spontaneous B cell lymphomas. Our findings indicate that MRN cannot be considered a standard tumor suppressor and instead imply that nuclease activities of MRE11 are required for oncogenesis. Inhibition of MRE11 nuclease activity increased DNA damage and selectively induced apoptosis in cells overexpressing oncogenes, suggesting MRE11 serves an important role in countering oncogene-induced replication stress. Thus, MRE11 may offer a target for cancer therapeutic development. More broadly, our work supports the idea that subtle enhancements of endogenous genome instability can exceed the tolerance of cancer cells and be exploited for therapeutic ends.

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IntroductionLiquid biopsies provide a non-invasive method to acquire the genetic information provided in cell-free DNA (cfDNA) as well as in single circulating tumour cells. Access to this genetic information through next-generation sequencing (NGS) identifies mutations and alterations such as Copy Number Variation (CNV) that play a role in cancer and other diseases.Material and methodsThe key to identifying rare mutations is improved sequencing accuracy and the ability to distinguish between biological and PCR duplicates. ThruPLEX Tag-seq was developed with the addition of unique molecular tags (UMTs) to improve sequencing accuracy by accounting for polymerase and sequencing errors and to increase confidence in rare allele identification. Whole Genome Amplification (WGA) for CNV detection was achieved by the thermal cycling quasi-random primed library chemistry of the PicoPLEX DNA-seq single-cell NGS library prep kit.Results and discussionsThruPLEX Tag-seq libraries were prepared using 10–30 ng of Horizon Discovery’s Multiplex I cfDNA Reference Standard Set containing six single nucleotide variants (SNV) for 4 different genes (EGFR, KRAS, NRAS, PIK3CA) present at 0.5%–5% allele frequency. The libraries were enriched with either a 110 kb or 240 kb custom panel or the Agilent ClearSeq Comprehensive Cancer Panel. Enriched libraries were sequenced witan average total read coverage of approximately 5,000X and analysed with and without the UMTs.For CNV analysis in single cells, a bar-coded PicoPLEX DNA-seq library was synthesised and amplified from 6 single cells from either PBMCs and clonally-expanded PC3 prostate cancer cells. Sequencing was performed on a MiSeq v2 and reads were mapped using BWA-MEM, processed in Picard_Mark_Duplicates, and further characterised in DNA nexus.classic. All PC3 cells showed reproducible CNV calling, however none of the lymphocyte samples showed any CNVs. Accurate CNV calls for PC3 cells were achieved even at when fastq files were randomly downsampled to 1 50 000 read pairs.ConclusionTherefore, use of UMTs in the preparation of NGS libraries from cfDNA enhances sequencing accuracy: by distinguishing between biological duplicates and PCR duplicates, increasing read coverage and decreasing background noise, reducing false positives, and in more confident mutation calls. PicoPLEX DNA-seq NGS libraries have a very simple and fast workflow that is suitable reproducible CNV detection in single cells even at low 0.002X average coverage.