Endogenous topoisomerase II-mediated DNA breaks drive thymic cancer predisposition linked to ATM deficiency

The ATM kinase is a master regulator of the DNA damage response to double-strand breaks (DSBs) and a well-established tumour suppressor whose loss is the cause of the neurodegenerative and cancer-prone syndrome Ataxia-Telangiectasia (A-T). A-T patients and Atm−/− mouse models are particularly predisposed to develop lymphoid cancers derived from deficient repair of RAG-induced DSBs during V(D)J recombination. Here, we unexpectedly find that specifically disturbing the repair of DSBs produced by DNA topoisomerase II (TOP2) by genetically removing the highly specialised repair enzyme TDP2 increases the incidence of thymic tumours in Atm−/− mice. Furthermore, we find that TOP2 strongly colocalizes with RAG, both genome-wide and at V(D)J recombination sites, resulting in an increased endogenous chromosomal fragility of these regions. Thus, our findings demonstrate a strong causal relationship between endogenous TOP2-induced DSBs and cancer development, confirming these lesions as major drivers of ATM-deficient lymphoid malignancies, and potentially other conditions and cancer types.

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For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub Other available datasets used in this study: RAG1 and RAG2 ChIPseq in thymocytes -SRA: PRJNA285688. Thymocytes ENDSeq -SRA: PRJNA326246. RAD21 ChIPseq in DP T-cells -SRA: PRJNA432324. POLR2A, H4K4me3, H3K27ac and CTCF -ENCODE: ENCSR000CEA, ENCSR000CCJ, ENCSR000CCH and ENCSR000CDZ respectively. Hi-C data in thymocytes -SRA: PRJNA435621.
Sample size was determined according to the minimal number of independent biological replicates that significantly identified an effect.
No data were excluded.
All experiments in this study were independently replicated, with biological and technical replicates as indicated in Figure Legends.
Animals were asigned an individual identification number (ID) upon birth. Animals of each genotype were randomly allocated to specific treatment conditions only considering their ID.
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Genome browser session (e.g. UCSC) Primary embryonic fibroblast and thymocytes were isolated from mice. n/a n/a n/a Wild-type, Tdp2-/-, Atm-/-and Tdp2-/-Atm-/-mice were generated by crossing Tdp2 +/-mice in mixed 129Ola x CD1 x C57BL/6 background and Atm+/-mice in mixed 129/SvEv x NIH Black Swiss background. Double heterozygous Tdp2+/-Atm+/-mice were selected and bred to maintain the colony and generate mice with the required genotypes.
Both male and females were used indistinctly.
For the analysis of lifespan and tumour incidence, animals were maintained throughout their life with a maximum of 2 years.
For individual treatment experiments, animals were used at 4 or 8 weeks of age, as indicated.