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

ARTICLE https://doi.org/10.1038/s41467-020-14638-w OPEN Endogenous topoisomerase II-mediated DNA breaks drive thymic cancer predisposition linked to ATM deficiency 1234567890():,; Alejandro Álvarez-Quilón 1,3,4, José Terrón-Bautista 1,4, Irene Delgado-Sainz1, Almudena Serrano-Benítez1, Rocío Romero-Granados1, Pedro Manuel Martínez-García1, Silvia Jimeno-González 1, Cristina Bernal-Lozano1, Cristina Quintero1, Lourdes García-Quintanilla1 & Felipe Cortés-Ledesma 1,2 ✉ 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. 1 Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), CSIC-Universidad de Sevilla-Universidad Pablo de Olavide, Sevilla 41092, Spain. 2 Topology and DNA breaks group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain. 3Present address: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada. 4These authors contributed equally: Alejandro Álvarez-Quilón, José Terrón-Bautista. ✉email: NATURE COMMUNICATIONS | (2020)11:910 | https://doi.org/10.1038/s41467-020-14638-w | www.nature.com/naturecommunications 1 ARTICLE A NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14638-w taxia Telangiectasia (A-T) is the paradigm of human inherited disease related to the DNA damage response (DDR)1,2. A-T is caused by loss-of-function mutations in the ATM gene, and is associated with multi-systemic features, affecting brain, gonads and the immune system; the main and most debilitating symptom of the disease being progressive earlyonset cerebellar ataxia. Spontaneous chromosomal instability and profound hypersensitivity to agents that induce DNA doublestrand breaks (DSBs) distinguish this syndrome from other spinocerebellar ataxias3, leading to the idea of DSBs underlying A-T symptomatology, although there is still an open debate regarding the molecular trigger of A-T, and the specific contribution of DSBs to the different aspects of the disease. The connection of DSBs with immunodeficiency and cancer predisposition in A-T is, however, well established and properly recapitulated in the Atm−/− mouse4. During lymphocyte development, the variable regions of T-cell receptor (TCR) and Immunoglobulin (Ig) loci, in T and B cells respectively, are randomly rearranged in a unique combination of the multiple possible V, D (in the case of TCRβ and δ and IgH) and J coding segments present in the germline5. This shuffling occurs through the generation of pairs of DSBs by the RAG1-RAG2 recombinase at recombination sequence signals (RSS), and their subsequent repair by the non-homologous end-joining (NHEJ) machinery. ATM, although not completely required, facilitates V(D)J recombination, providing a molecular explanation for the relatively mild immunodeficiency characteristic of A-T patients and Atm−/− mice. The main role of ATM in V(D)J recombination is to stabilize RAG post-cleavage complexes, thus protecting DNA ends and promoting their correct use for ligation6,7, but other functions that are not fully understood become relevant in conditions in which NHEJ is compromised8–10. Defects in V(D)J recombination are not only responsible for the immunological problems in A-T, but also underlie its characteristic cancer predisposition. Indeed, roughly one third of patients develop cancer, mainly lymphoma or lymphocytic leukemia, which in mice manifests as very aggressive thymic neoplasias, with characteristics of T-cell acute lymphoblastic leukemia (T-ALL)4. A-T and Atm−/− T-cell malignancies are frequently linked to genome rearrangements involving the TCR loci, strongly supporting the contribution of aberrant V(D)J recombination. Most prevalent in A-T are translocations or inversions (14;14) involving the TCRα/δ locus. Interestingly, this event is also frequent in mice as a t(14;12) translocation, providing thus a molecular link between ATM-deficient T-cell malignancies in human and mouse11–14. Furthermore, T-cell malignancies in Atm−/− mice and human share other characteristics such as chromosome 15 duplication, Notch1 amplification and Pten deletion13,14. In the currently accepted model, deficient V(D)J recombination and checkpoint defects caused by ATM loss lead to persistent DSBs that engage in oncogenic rearrangements15. Aberrant V(D)J recombination, however, is unlikely to represent the single driver of oncogenic translocations, as evidenced by additional V(D)J-unrelated regions of instability, and the persistent cancer predisposition observed upon RAG deficiency16,17. These results strongly suggest additional sources of DSBs as relevant contributors to the ATM-deficient oncogenic translocations responsible for T-cell cancer predisposition. In this sense, aberrant action of DNA topoisomerase II (TOP2) can constitute an important source of chromosomal breakage18. The physiological function of TOP2 is to solve topological problems arising from DNA metabolism19. To do so, it cleaves both strands of DNA to gate the passage of another DNA segment, via the formation of a catalytic intermediate, the cleavage complex (TOP2cc), in which the enzyme remains covalently linked to 5′ termini of the break. Although normally 2 very transient, as the DNA gate is rapidly resealed after DNA passage, TOP2ccs can be stabilized and result in irreversible TOP2-induced DSBs upon conflict with cellular processes and processing of the structure by the proteasome20. Interestingly, this does not only occur accidentally as a consequence of errors in TOP2 catalytic cycles, but underlies the clinical efficacy of a heterogeneous group of chemotherapeutic agents collectively known as TOP2 “poisons”, of which etoposide is a widely characterised paradigmatic example21. Despite substantial efficacy, treatment with TOP2 poisons has been traditionally linked to the development of secondary haematological malignancies characterized by specific translocations22. In this regard, transcription and loop extrusion, a process that organizes the (...truncated)