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

ARTICLE Endogenous topoisomerase II-mediated DNA breaks drive thymic cancer predisposition linked to ATM deficiency 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. https://doi.org/10.1038/s41467-020-14638-w OPEN 1Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), CSIC-Universidad de Sevilla-Universidad Pablo de Olavide, Sevilla 41092, Spain. 2Topology and DNA breaks group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain. 3 Present address: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada. 4 These authors contributed equally: Alejandro Álvarez-Quilón, José Terrón-Bautista. ✉email: [email protected] NATURE COMMUNICATIONS | (2020) 11:910 | https://doi.org/10.1038/s41467-020-14638-w |www.nature.com/naturecommunications 1 1234567890():,; Ataxia 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 TCRβand δand 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/δlocus. 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.Todoso, 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 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 genome in functional loop domains by threading chromatin through the ring-shaped cohesin complex23–25, have been proposed as the main source of DSBs and chromosomal translocations induced by etoposide treatment26–29. However, the incidence and impact of endogenous TOP2-mediated lesions in the absence of treatment with TOP2 poisons and their relationship with cancer development remain to be established. Concerning this, compounds present in the diet and the environment and some forms of pre-existing DNA damage, such as nicks or abasic sites, can also poison TOP2 activity18. Remarkably, TOP2-induced DSBs are characterized by harbouring peptide blocks derived from the enzyme that remain covalently bound to 5′termini through a phosphotyrosyl bond. TDP2, with its 5′-tyrosyl-DNA-phosphodiesterese activity30,31,is the only mammalian enzyme able to directly unblock TOP2- induced DSBs for their repair32. Interestingly, we have previously reported that ATM is specifically involved in facilitating repair of TOP2-induced DSBs in a TDP2-independent fashion, probably by promoting alternative nucleolytic pathways for the unspecific removal of the peptide adducts33. TDP2 and ATM, therefore, define two independent pathways for the repair of TOP2-induced DSBs, the combined absence of which has dramatic consequences for the cellular response to these lesions. In the present study, we generate and characterise Atm−/− Tdp2−/−double-deficient mice to address the occurrence and relevance of TOP2-mediated DSBs in vivo, and, most importantly, their possible contribution to the different aspects of A-T symptomatology. We find that TDP2 loss further aggravates the predisposition of Atm−/−mice to thymic malignancies, and show a strong colocalization between RAG and TOP2 at regions that endogenously accumulate DSBs and TOP2ccs in Atm−/−Tdp2−/− thymocytes. These results strongly suggest the co-occurrence and misrepair of RAG- and TOP2-mediated DSBs as a major driving force of thymic malignancies linked to ATM deficiency. Results Tdp2−/−Atm−/−and Atm−/−mice are phenotypically similar. Considering the strong sensitivity and repair defect of Tdp2−/− Atm−/−double-deficient cells to TOP2-induced DSBs33,we decided to address the physiological impact of these lesions in the context of the entire organism. We reasoned that, if TOP2 damage is mediating deleterious effects in the Atm−/−mouse, these would be further aggravated by Tdp2 loss. Mice from Tdp2 and Atm double heterozygote crosses were born at the expected Mendelian proportions and not displaying gross abnormalities (Supplementary Fig. 1a). Since Atm loss causes marked growth retardation4, we measured body weight of male mice at 4 weeks of age (Supplementary Fig.1b). As expected, Atm−/−mice showed a more than 20% reduction in size compared to wild-type animals. Deletion of Tdp2, however, did not have an impact on its own, as previously reported32, neither did it aggravate the growth retardation of Atm−/−mice. Furthermore, neither Tdp2−/−Atm−/− ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14638-w 2NATURE COMMUNICATIONS | (2020) 11:910 | https://doi.org/10.1038/s41467-020-14638-w |www.nature.com/naturecommunications nor, as previously reported4,Atm−/−mice displayed clear symptoms of neurological deficiencies or an ataxic behaviour, which we confirmed by analysing cerebellar integrity and Purkinje cellular density in 8-week old mice (Supplementary Fig. 1c). Finally, Tdp2−/−mutation did not substantially modify the already strong disruption of spermatogenesis found in Atm−/− mice (Supplementary Fig. 1d). Primary Tdp2−/−Atm−/−mouse embryonic fibroblasts (MEFs), however, showed a marked reduction in proliferation compared to single-mutant and wild-type cells (Supplementary Fig. 1e), and spontaneously accumulated DSBs in unchallenged growth conditions (Fig. 1a), with 60% of Tdp2−/−Atm−/−cells harbouring more than one 53BP1 focus compared to 25% in wildtype. Overall, these results suggest an accumulation of endogenous DSBs in the absence of TDP2 and ATM which, at least under certain growth conditions, can disrupt cellular fitness. The in vivo incidence of these lesions, however, does not seem sufficiently high to seriously compromise tissue homeostasis during development and early stages of life. Tdp2−/−Atm−/−mice are hypersensitive to TOP2-induced DSBs. Given the results above, we decided to challenge Tdp2−/− Atm−/−mice with an increased load of TOP2-induced lesions. To do so, 8-week old adult mice of the relevant genotypes were subjected to a single intraperitoneal injection of the TOP2-poison etoposide (25 mg/kg). As can be seen in Fig. 1b, Tdp2−/−Atm−/− double-knockout mice showed severe hypersensitivity to etoposide exposure, suffering a progressive weight loss that reached, on average, a 15% decrease after 6 days. Histopathological analysis revealed marked villous atrophy in the small intestine mucosa, which is a known etoposide target, as the likely cause of the drastic weight loss in Tdp2−/−Atm−/−(Fig. 1b). In contrast, at this low etoposide concentration, wild-type and Atm−/−animals did not respond negatively to the treatment, while Tdp2−/−did so only mildly. This synergistic hypersensitivity of Tdp2−/−Atm−/− mice to etoposide suggests a protective role of ATM against the adverse effects of TOP2 lesions in vivo, but only when TDP2 is absent, which recapitulates cellular observations in the context of the entire organism33. TDP2 loss increases thymic cancer predisposition of Atm−/−. We decided to analyse Tdp2−/−Atm−/−mice over a longer period, to allow an accumulation of a higher load of TOP2-mediated damage and/or the development of potential pathologies. For this, weight and general health status were monitored weekly in a minimum of 20 mice for each of the relevant genotypes, until reaching the experimental endpoint of 2 years (730 days) (Fig. 2a). As previously reported, we observed that Atm−/−mice showed a reduced life-span with an important number of animals succumbing to thymic neoplasia after the first few months of life (Fig. 2b, Supplementary Fig. 2a). Furthermore, a molecular characterization of the cellular content in these tumours showed a 0123456 70 80 90 100 Tdp2+Atm+/+ Tdp2–/–Atm+/+ Tdp2+Atm–/– Tdp2–/–Atm–/– P < 0.0001 % of original weight Time after injection (days) a b Tdp2 +/+ Tdp2 –/– Atm+/+ Atm–/– 20 µm P = 0.0208 P = ns P = ns Tdp2 Atm ++ ++ –– –– % MEFs > 1 53BP1 focus 0 20 40 60 80 Tdp2+ Atm+/+ Tdp2+ Atm–/– Tdp2–/– Atm+/+ Tdp2–/– Atm+/+ 500 µM Fig. 1 Tdp2−/−Atm−/−causes the spontaneous accumulation of DSBs and etoposide hypersensitivity in mice. a Endogenous DSB occurrence in Tdp2+/+Atm+/+,Tdp2 −/−Atm+/+,Tdp2 +/+Atm−/−and Tdp2−/−Atm−/−primary MEFs measured by 53BP1 foci formation. The percentage of cells with more than one focus (left) and representative images (right) of 53BP1 foci (green) and DAPI staining (blue) are shown for each genotype. MEFs isolated from three different embryos were analysed per condition. Mean ± SEM and statistical significance by one-way ANOVA with Bonferroni post-test is shown (F=5,402). bBody weight of 8-week old mice cohorts intraperitoneally injected with a single dose of etoposide (25 mg/kg). Average ± SEM of the percentage of initial body weight from five mice and statistical significance by Two-way ANOVA with Bonferroni post-test is shown (F=29,11). Tdp2+ indicates both Tdp2+/+and Tdp2+/−genotypes. Representative images of Haematoxylin-Eosin stained jejunum slices obtained 6 days after etoposide exposure are shown. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14638-w ARTICLE NATURE COMMUNICATIONS | (2020) 11:910 | https://doi.org/10.1038/s41467-020-14638-w |www.nature.com/naturecommunications 3 majority (70%) of double positive CD4+CD8+T-cell tumours (Fig. 2c, Supplementary Fig. 2b), consistent with the reported T-ALL predisposition. Single Tdp2−/−mice had a lifespan comparable to wild-type (Fig. 2a), and none of the animals developed thymic lymphoma within the course of the experiment (2 years). Strikingly however, in Atm−/−background, both the reducedoveralllife-spanandthe incidence of thymic tumours were largely aggravated by Tdp2 inactivation (Fig. 2a, b). Thus, Tdp2−/− Atm−/−mice showed a median survival of 140 days, contrasting with 307 days for the Atm−/−single mutant (Fig. 2a), and the probability of developing a thymic tumour during the 1st year of life increased from a 43% in Atm−/−to a 72% in the Tdp2−/− Atm−/−double mutant (Fig. 2b). Importantly, lifespan of Tdp2−/− Atm−/−and Atm−/−mice that did develop a thymic tumour was not significantly different (Fig. 2d), suggesting a direct effect on the incidence and not on the latency or aggressiveness of T-cell malignancies. In this sense, tumours observed in Tdp2−/− Atm−/− mice were also predominantly formed by double positive CD4+CD8+T-cell precursors (80%) (Fig. 2c). Doubleknockout mice, however, did not show differential distribution in the populations of double negative (CD4−CD8−), double positive (CD4+CD8+) or single positives (CD4+or CD8+)thymocytes compared to Atm−/−mice (Supplementary Fig. 2c), suggesting that the increased incidence of T-cell malignancy caused by TDP2 loss is unlikely to reflect further disruption of V(D)J recombination. In the same way, pre-B-cell, pro-B-cell and immature B-cell populations did not differ between single Atm−/−and double Tdp2−/− Atm−/−knock-out mice (Supplementary Fig. 2d), further supporting the idea that V(D)J recombination is not substantially altered by TDP2 loss. The checkpoint and proapoptotic functions of ATM, which are strongly signalled through the p53-dependent pathway, are 0 150 300 450 600 750 0 25 50 75 100 a c ef d b Tdp2+ Atm+/+ Tdp2–/– Atm+/+ Tdp2+ Atm–/– Tdp2–/– Atm–/– Tdp2+ Atm–/– Tdp2–/– Atm–/– Tdp2–/– Atm–/– Time (days) Percent survival (%) P = 0.0481 Trp53 –/– Tdp2 +/+ Trp53 –/– Tdp2 –/– 0 50 100 150 200 250 0 20 40 60 80 100 Trp53 –/– Tdp2 +/+ Trp53 –/– Tdp2 –/– Time (days) Percent survival (%) 0 100 200 300 400 0 20 40 60 80 P = 0.0123 Time (days) 0 200 400 600 Median survival (days) 0 50 100 150 200 250 0 20 40 60 80 Time (days) Double positive Single positive 0 20 40 60 80 100 Thymic lymphoma (%) Tdp2+ Tdp2–/– Atm–/– Tdp2+ Cumulative occurrence of thymic lymphoma (%) Cumulative occurrence of thymic lymphoma (%) Fig. 2 TDP2 deficiency increases thymic cancer predisposition of Atm−/−but not of Tpr53−/−mice. a,bKaplan–Meier survival curve (a) and cumulative occurrence (b) of thymic tumours during the 1st year of life of at least 20 mice with the indicated Tdp2 and Atm genotypes. n=21 (Tdp2+/+ Atm+/+),n=23 (Tdp2−/−Atm+/+),n=23 (Tdp2+/+Atm−/−), n=21 (Tdp2−/−Atm−/−). Tdp2+indicates both Tdp2+/+and Tdp2+/−genotypes. Statistical significance by two-sided Wilcoxon test is indicated. cPercentage of tumours composed by immature CD4+CD8+double positive, or CD4+/CD8+single positive lymphocytes in the indicated genotypes. Thymocytes were selected by size and complexity prior classification by high or low CD4 and CD8 markers (see Supplementary Fig. 1a for gating strategy). dMedian life-span of Tdp2+/+Atm−/−and Tdp2−/−Atm−/−mice affected by a thymic tumour (n=11 and n=16 for Tdp2+/+Atm−/−and Tdp2−/−Atm−/−mice respectively). eKaplan–Meier survival curve and fcumulative incidence of thymic tumours of the indicated Tdp2 and Trp53 genotypes (n=16 for Tdp2+/+and n=22 for Tdp2−/−). ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14638-w 4NATURE COMMUNICATIONS | (2020) 11:910 | https://doi.org/10.1038/s41467-020-14638-w |www.nature.com/naturecommunications known to be determinant for Atm−/−cancer predisposition15. Consistent with this, p53-deficient Trp53−/−mice are also highly susceptible to the development of double positive CD4+CD8+ thymic tumours34. In order to discern between the contribution of repair and checkpoint/apoptotic functions of ATM in the increased incidence of thymic tumours in Tdp2−/−Atm−/−,we analysed life span and tumour incidence in Tdp2−/−Trp53−/− animals, in which the repair component is not substantially affected. In contrast to what was observed in Atm−/−background (Fig. 2a, b), loss of functional TDP2 did not significantly decrease lifespan (Fig. 2e), nor increase the high incidence of cancer (Supplementary Fig. 2e), and thymic lymphoma in particular (Fig. 2f), observed in Trp53−/−animals. This suggests that Tdp2−/−Atm−/−tumours likely reflect direct functions of ATM in the repair of TOP2-induced DSBs, and not only in their signalling for checkpoint and apoptosis. Overall, the results are consistent with a model in which misrepair of TOP2-induced DSBs can strongly contribute to the development of thymic tumours characteristic of ATM deficiency. Tdp2−/−Atm−/−and Atm−/−malignancies are molecularly similar. In order to gain insights into the molecular events responsible for thymic tumour development in Tdp2−/− Atm−/− mice, we analysed and compared copy-number variation by comparative genomic hybridization (CGH) in six Tdp2−/− Atm−/−and three Atm−/−thymic tumours (Fig. 3a, Supplementary Fig. 3 and Supplementary Table 1). In Atm−/−animals (Fig. 3a, b), we observed the previously reported features of genomic instability13,withamplification upstream of the Tcra/d locus in chromosome 14 (2 out of 3 mice) and variable hemizygous loss of a Bcl11b-containing telomeric region of chromosome 12 (all 3 mice), which are indicative of the frequent t(14;12) translocation and subsequent breakage-fusion-bridge cycles. In addition, one mouse displayed instability at the Tcrb locus, with both gain and loss of DNA sequence. Two of the three animals also presented the characteristic duplication of chromosome 15. Also, two animals presented deletions in chromosome 19 covering the Pten tumour suppressor, although only in one of the cases this corresponded to the previously reported homozygous loss13. Finally, we did not observe amplification of Notch1 in chromosome 2, which is another common, although less frequent, feature in Atm−/−T-cell malignancies. Surprisingly, we observed very similar features in Tdp2−/− Atm−/− mice (Fig. 3a, b). Four out of six tumours presented Tcra/d linked amplification, and all six showed chromosome 12 deletion with Bcl11b loss. Instability at the Tcrb locus was also observed in two of the six cases. Trisomy of chromosome 15 was also observed in four of the six animals, while only one of the six cases carried Notch1 amplification. All these events were coincident with the results obtained in Atm−/−animals (see above) and previous reports13. Finally, as observed for Atm−/−,oneTdp2−/−Atm−/− animal displayed hemizygous Pten loss as part of a larger deletion. The variable length of the copy-number variation events at common regions, together with additional sites of instability that are unique to individual tumours (Supplementary Fig. 3), are indicative of the stochastic nature of the oncogenic events. In summary, we can conclude that loss of TDP2 increases the incidence of the same oncogenic rearrangements that drive thymic tumour in Atm−/−animals, suggesting a strong contribution of TOP2-mediated lesions to ATM-deficient T-cell cancer predisposition. TOP2B is enriched at sites of DSB accumulation. In order to further establish aberrant TOP2 activity as a direct contributor to Atm−/−oncogenic T-cell genome rearrangements, we decided to map binding of topoisomerase IIß (TOP2B), which is the main source of TOP2 activity in G1 and non-cycling cells. ChIPseq analysis in freshly isolated thymocytes from wild-type mice revealed a pattern of TOP2B distribution consistent with that previously found in other cell types and tissues35–37 (Fig. 4a), with a clear enrichment at promoter, enhancer and insulator regions (34%, 19% and 13% of TOP2B peaks, respectively). In fact, regardless of the type of functional element analysed, and as previously reported36, we observed a strong correlation between TOP2B and the core cohesin subunit RAD21, with 81% of TOP2B peaks overlapping with those of cohesin (Fig. 4b), and detectable cohesin signal in virtually all TOP2B peaks, regardless of the presence of RNA polymerase II (POLR2A) or the insulating architectural protein CTCF (Fig. 4c). Fig. 4d shows the Cxcr4 locus merely as an illustrative example of TOP2B and cohesin co-localization within promoter (left) and insulator (right) regions. These results are consistent with the proposed connection between TOP2B function and 3D-genome organisation36,37. We decided to specifically study TOP2B occupancy at particular sites related to Atm−/−oncogenic translocations, integrating published maps of endogenous DSB accumulation determined by ENDseq in Atm−/−thymocytes 38. When concentrated on the Bcl11b,Notch1, and Pten loci (Fig. 5a–c), we observed clear TOP2B binding at all of these regions with strong accumulation at the promoters and towards the 3’end of the genes, with a pattern following that of cohesin and RNA- polymerase II (POLR2A). This TOP2B accumulation was not thymocyte-specific, as a similar pattern was observed in MEFs (shown for Bcl11b, Supplementary Fig. 4a). In order to directly identify regions of DSB accumulation in Atm−/−thymocytes, we selected ENDseq peaks specific for Atm−/−thymocytes (highlighted in yellow; Fig. 5a–c). Interestingly, when these sites were compared with the distribution of TOP2B, overlap was highly variable with examples of both unique and common regions; compare region 1 and region 2 in Pten (Fig. 5c). TOP2B is therefore present at Atm−/−unstable regions, but it is difficult to establish a direct association with DSB accumulation, and may be a mere consequence of its widespread distribution at regulatory regions. Furthermore, the difficulty to assign original translocation breakpoints with sufficient precision strongly limits our capacity to directly link TOP2B binding to oncogenic DSB occurrence at these specific locations. Based on this, we decided to compare the distribution of TOP2B and ENDseq signal at a genome-wide level. We observed a clear accumulation of DSBs at TOP2B peaks (Fig. 5d), supportive of a link between TOP2B function and spontaneous chromosome fragility, in the same line as what has been previously reported for etoposide treatments37.Conversely, and further supportive of this TOP2B-DSB association, ENDseq peaks displayed an accumulation of TOP2B signal (Fig. 5e). Most prominent peaks of ENDseq signal in thymocytes are known to be associated with RSS sites, disappearing in Rag2−/−and strongly accumulating in Atm−/− cells38, consistent with the high incidence and precision of RAG cleavage and the reported roles of ATM in facilitating V(D)J recombination. At TOP2B sites, however, DSBs were noticeably increased in both Rag2−/−and Atm−/−thymocytes (Fig. 5d), demonstrating that they are unrelated to RAG cleavage, and suggesting that these lesions can accumulate upon ATM deficiency. In summary, there is a significant genome-wide correlation between sites of TOP2B function and occurrence of RAG-independent endogenous DSBs in thymocytes, suggesting aberrant TOP2B activity as an additional relevant source of chromosome breakage that could contribute to characteristic Atm−/−oncogenic translocations. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14638-w ARTICLE NATURE COMMUNICATIONS | (2020) 11:910 | https://doi.org/10.1038/s41467-020-14638-w |www.nature.com/naturecommunications 5 TOP2B and cohesin colocalize with the RAG endonuclease. Based on the results above, we decided to check how RAG and TOP2B contribute to DSB accumulation in more detail. In order to do so, we analysed TOP2B, RAD21 and ENDseq signal in previously reported peaks of RAG1 and RAG212 (Fig. 6a–c). RAG2 has a wide distribution with a strong presence at active promoters displaying the H3K4me3 histone mark, while the presence of RAG1 is more restricted, only co-localizing with a fraction of RAG2 peaks39,40. We observed a striking genome-wide co-localization of TOP2B and RAD21 with RAG2, which was irrespective of the strong presence of RAG1 or not (Fig. 6a). Thus, clear TOP2B accumulation was observed in peaks containing both RAG1 and RAG2 but also in those containing mostly RAG2 (Fig. 6a, b). In addition, 42% of TOP2B peaks colocalized with RAG (merge of RAG1 and RAG2 peaks) (Fig. 6b), consistent with their mutual enrichment at H3K4me3-positive promoter regions. In any case, although some ENDseq signal was observed at these regions, the accumulation of DSBs was not clear or sufficiently localized (Fig. 6a). Interestingly, when directly compared, spontaneous DSB accumulation was more robust at TOP2B and cohesin than at RAG1 or RAG2 peaks, both in wild-type and Atm−/−thymocytes (Fig. 6d). Altogether, these results uncover a surprising genome-wide connection between RAG and TOP2B- cohesin, and suggest that, when analysed globally, TOP2B activity can constitute an important source of DNA breakage in Atm−/− thymocytes, quantitatively more relevant than DSBs directly induced by RAG. Based on this genome-wide colocalization between TOP2B and RAG, we decided to check the distribution of TOP2B specifically at sites actively undergoing V(D)J recombination in thymocytes (Fig. 7a–c; Supplementary Fig. 5a, b); this includes the Tcra/d, Tcrb,Tcrg and IgH15 loci. Strikingly, sites of strong and localized −3 −2 −1 0 1 2 3 a b Avge log2 (tumor/kidney) chr: Tcrg Notch1 Igh Tcl1 Bcl11b Tcra/d Pten 12345678910111213141516171819 −3 −2 −1 0 1 2 3 Avge log2 (tumor/kidney) chr: Tcrg Notch1 Igh Tcl1 Bcl11b Tcra/d Pten 1 2 3 4 5 6 7 8 9 10 1112 13141516171819 Tcrb Tcrb Tdp2 +/+ Atm –/– Tdp2 –/– Atm –/– Tdp2 +/+ Atm –/– Tdp2 –/– Atm –/– Genotype Amp (Tcra/d) 2/3 4/6 Del (12) 3/3 6/6 Del (Tcrb) 1/3 2/6 Amp (15) 2/3 4/6 Del (Pten) 2/3 1/6 Amp (Notch1) 0/3 1/6 Fig. 3 Tdp2−/−Atm−/−and Atm−/−thymic malignancies display similar genome rearrangements. a Merged CGH analysis of Tdp2+/+Atm−/−(three mice, top) and Tdp2−/−Atm−/−(six mice, bottom) thymic lymphomas. DNA from each tumour sample was hybridized and analysed using kidney DNA from the same mouse as a control. Average amplification (red) or deletion (blue) score (Log2 tumour/kidney ratio) is shown. Significant copy number variations are defined by −0.66>Log2 tumour/kidney>0.66 (dashed lines). The location of relevant loci in Atm−/−thymic tumours is indicated. bTable summarising the number of mice of each genotype displaying copy number variation at the indicated locus of interest. Amp(Tcra/d): amplification upstream of Tcra/d; Del(12): deletion of the telomeric region of chromosome 12 covering Bcl11b; Del(Tcrb): deletion at the Tcrb locus; Amp(15): trisomy of chromosome 15; Del(Pten): deletion at the Pten locus; Amp(Notch1): duplication at the Notch1 locus. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14638-w 6NATURE COMMUNICATIONS | (2020) 11:910 | https://doi.org/10.1038/s41467-020-14638-w |www.nature.com/naturecommunications TOP2B accumulation were observed at all of these sites, and were particularly enriched at the V(D)J-initiating J segments. As expected, TOP2B coincided with the position of the cohesin subunit RAD21, and, in line with the genome-wide observations, also with the strongest peaks of RAG1 and RAG2. Furthermore, the TOP2B-cohesin sites concurred with a very strong ENDseq signal that was further enhanced in Atm−/−thymocytes. Interestingly however, DSB accumulation in these regions was completely dependent on RAG, and did not fully coincide with TOP2B-cohesin peaks, but was displaced a few kbs from TOP2B sites (Supplementary Fig. 6). This demonstrates that direct RAG cleavage, rather than aberrant TOP2B activity, is the main source of DSBs at Tcr and Igh loci in thymocytes, but suggests a strong association between TOP2B-cohesin function and sites undergoing active V(D)J recombination. In support of this, TOP2B accumulation in these regions was specific to thymocytes when compared to peaks called in MEFs (Supplementary Fig. 4b, c). ChIPseq of TOP2A also evidenced that TOP2 enrichment was Promoters 34% Insulators 13% Enhancers 19% 1% 1% 2% TOP2B peaks Genome a 3 4 5 6 7 −5 kb 0 bp 5 kb Promoters Enhancers Insulators Promoters Enhancers Insulators 100 kb 130,500,000 130,550,000 130,600,000 130,650,000 130,700,000 Cxcr4 16.2 2 _ 109.2 6.51 0.19 _ 3.41 0.10 _ 27.46 0.12 _ 12.59 0.14 _ TOP2B RAD21 CTCF POLR2A H3K4me3 H3K27ac d RAD21 26664 TOP2B 1003 81% b RAD21 CTCFPOLR2ATOP2B 1003 1 −5 kb 0 bp 5 kb 4 8 12 16 C2 C1 1003 1 −5 kb 0 bp 5 kb 1234567 C2 C1 1003 1 −5 kb 0 bp 5 kb 0 50 150 250 C2 C1 1003 1 −5 kb 0 bp 5 kb 2468 C2 C1 c TOP2B IgG Fig. 4 TOP2B colocalizes with cohesin at promoter, enhancer and insulator regions in thymocytes. a Genome-wide distribution of 1003 TOP2B ChIPseq called peaks at different genomic features (promoters, enhancers, insulators and others) in wild-type primary thymocytes. Chromatin states were defined using ENCODE ccREs registry: H3K4me3, TSS and low H3K27ac for promoters, H3K27ac and low H3K4me3 for enhancers and high CTCF and no presence of the previous histone marks for insulator-like regions.TOP2B peak distribution compared to the entire genome (top), and global signal profile compared to IgG control (bottom) at each genomic feature is shown. TOP2B peaks were defined as common peaks between TOP2B ChIPseq experiments using two different antibodies. bOverlap of TOP2B and RAD21 peaks in wild-type thymocytes. cGenome browser view of TOP2B, RAD21, CTCF, POLR2A, H3K4me3 (ENCODE) and H3K27ac (ENCODE) signal tracks at a representative genomic region encompassing an active promoter (left, positive for POLR2A, H3K4me3 and H3K27ac) and an insulator (right, negative for POLR2A, H3K4me3 and H3K27ac, and positive for CTCF and RAD21). Sites highlighted in grey show sites of major TOP2B signal accumulation over the region. dHeatmaps of TOP2B (this study), POLR2A (ENCODE), RAD2147 and CTCF (ENCODE) ChIPseq signals at TOP2B peaks (± 5 kb). K-means clusters based on the different signals represented are shown divided by black lines and named as C1 and C2. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14638-w ARTICLE NATURE COMMUNICATIONS | (2020) 11:910 | https://doi.org/10.1038/s41467-020-14638-w |www.nature.com/naturecommunications 7 preferential for the TOP2B isoform, both at V(D)J recombination sites and other genes related with the observed oncogenic translocations (Supplementary Fig. 4). Furthermore, when END- seq signal at Tcrb was analysed in detail, there were two peaks that became apparent when the predominant signal derived from RAG cleavage was lost in Rag2−/−thymocytes (Fig. 7c; ENDseq Rag2−/−autoscaled). This relocation of ENDseq signal from major sites of RAG cleavage to additional less prominent sites may be responsible for the apparent increase in DSBs observed at TOP2B peaks in Rag2−/−background (Figs. 5a and 6d). In any case, similarly to what occurred on a genome-wide scale and at other particular locations (Fig. 5), these regions of DSB accumulation were perfectly coincident with the two major sites of TOP2B and RAD21 binding, further supporting an association between TOP2B function and RAG-independent DNA breakage. Nevertheless, despite a similar localized accumulation of TOP2B- cohesin, RAG-independent ENDseq signal was below detection levels at other Tcr and Igh loci (Fig. 7a, b; Supplementary Fig. 5), indicating some degree of variability in the incidence and/or detection of TOP2B-mediated DSBs at these regions. Tdp2−/−Atm−/−thymocytes accumulate endogenous TOP2 DSBs. To directly confirm aberrant TOP2B activity as a relevant source of DSB accumulation in Tdp2−/−Atm−/−thymocytes, we used a technique recently developed in the laboratory, ICE-IP, in which TOP2 covalently attached to DNA is immunoprecipitated, and the abundance of the genomic region of interest is measured by qPCR (see the “Methods”section and Supplementary Fig. 7 for a detailed description). This allows locus-specific detection of TOP2ccs and TOP2-induced DSBs in which TOP2 has not been fully degraded or removed. In order to be able to detect endogenous lesions, we included a step of nested amplification. We first concentrated on two regions of Tcrb in which RAG- independent ENDseq peaks were detected (region 1 and region 2 in Fig. 7c). Interestingly, TOP2B ICE-IP in freshly isolated thymocytes showed a very clear enrichment of covalently attached TOP2B, exclusively, or at least only significantly, in Tdp2−/−Atm−/−thymocytes, with a >10-fold increase compared to wild-type animals (Fig. 7d). We then tested two regions of Pten with Atm−/−-specific ENDseq signal, one that coincided with a strong TOP2B signal and one that did not (region 1 and region 2 in Fig. 5c, respectively). As expected, covalently attached TOP2B accumulated to significant levels only in region 1 of Pten in Tdp2−/−Atm−/−thymocytes, and neither in region 2, nor in wild-type or single-mutant cells. These results strongly support the idea that TDP2 and ATM independently operate to prevent the endogenous accumulation of TOP2B-blocked DSBs, both 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 −5 kb 0 bp 5 kb WT Atm–/– Rag2 –/– WT TOP2B at ENDseq p eaks ab 50 kb 32,850,000 32,900,000 9.15 _ 0.19 _ 10 _ 0 _ 10 _ 0 _ 17 _ 1.6 _ 71.315 _ 0.96 _ Pten RAD21 ENDSeq WT ENDSeq Atm–/– TOP2B POLR2A Bcl11b RAD21 ENDSeq WT ENDSeq Atm–/– TOP2B POLR2A d c RAD21 ENDSeq WT ENDSeq Atm–/– TOP2B POLR2A Notch1 20 kb 26,315,000 26,335,000 26,355,000 14.5 _ 2 _ 11.89 _ 0.19 _ 82.48 _ 0 _ 10 _ 0 _ 10 _ 0 _ 2.6 2.8 3.0 3.2 3.4 3.6 −5 kb 0 bp 5 kb ENDseq Input e ChIPseq signal (RPKM) ENDseq signal (RPKM) 109,150,000 109,250,000 100 kb 20 _ 2 _ 4.47 _ 0.25 _ 142 _ 0 _ 5 _ 0 _ 5 _ 0 _ Region 1 Region 2 Fig. 5 TOP2B colocalizes with endogenous DSB accumulation in thymocytes. a–cGenome browser view of TOP2B, POLR2A, RAD21, and ENDseq (DSBs) signal tracks in wild-type and Atm−/−primary thymocytes, as indicated, at Bcl11b (a), Notch1 (b) and Pten (c) loci. Atm−/−-specific ENDseq peaks, defined as those not called in wild-type thymocytes, are highlighted in yellow. Two regions of interest in Pten are indicated as region 1 and region 2. dGlobal profile of ENDseq signal at TOP2B peaks in wild-type, Atm−/−and Rag2−/−primary thymocytes. Control input signal is also shown. eGlobal profile of TOP2B signal at ENDseq peaks in wild-type primary thymocytes. Set of ENDseq peaks were determined as the merge between those detected in any of the analysed genetic conditions. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14638-w 8NATURE COMMUNICATIONS | (2020) 11:910 | https://doi.org/10.1038/s41467-020-14638-w |www.nature.com/naturecommunications globally at particular regions throughout the genome, and specifically at sites undergoing V(D)J recombination. Finally, in order to reinforce the idea that the rearrangements found in thymic lymphomas could be related to 3D-genome folding and the functions of TOP2B in this process, available Hi–C maps on wild-type thymocytes 41 were integrated with hotspots of Atm−/−-linked genomic instability. As a matter of fact, V(D)J recombination sites and other unstable sites were clearly associated with regions of strong long-range interactions (in the order of megabases), apparent as borders of squares in the contact density map (Supplementary Fig. 8), and that could be associated to topologically associating domains (TADs) borders. Interestingly, these regions have been shown to be particularly prone to breakage and instability in general 42,43, and to the accumulation of etoposide-induced DSBs in particular37, which has led to the idea of TOP2 activity at these functional elements of genome organization as a potential major driver of oncogenic translocations28,29. Discussion In this study, we show that the absence of TDP2 function significantly aggravates the predisposition of Atm−/−mice to develop thymic malignancies (Fig. 2). Furthermore, TOP2B genome-wide binding correlates with sites of endogenous DSB accumulation (Fig. 5), and endogenous TOP2B-blocked lesions indeed accumulate in Tdp2−/−Atm−/−thymocytes (Fig. 7). These observations strongly support TOP2B-mediated DSBs as potential drivers of tumour development, at least in the particular setting of A-T-linked cancer predisposition, establishing a causal connection between misrepair of endogenous TOP2 lesions and tumorigenesis. In this sense, the implications of these results reach beyond our understanding of A-T cancer predisposition, since ATM is a well-established tumour suppressor, very frequently mutated in in many tumour types, and lymphoid malignancies in particular44. Interestingly, thymic tumours in Tdp2−/−Atm−/−mice display identical characteristics to those of Atm−/−animals, with a very aggressive form of T-ALL associated to recurrent clonal genome rearrangements enriched at loci undergoing V(D)J recombination (Figs. 2–3; Supplementary Fig. 2–3 and Supplementary Table 1). This is, in principle, difficult to reconcile with the incidence of thymic malignancies being increased by loss of TDP2, an enzyme that is highly specialized in TOP2-mediated DSBs, and therefore completely unnecessary for the repair of hairpin DNA ends produced by RAG. Nevertheless, one must bear in mind that translocations require two DSBs, so that TOP2- mediated lesions can contribute to the oncogenic events by providing partners for rearrangements that also engage Tcr or Ig loci, being the co-occurrence of TOP2- and RAG-mediated DNA breaks, and their stabilization and misrepair upon ATM loss, and not RAG-induced breaks per se, what drives ATM-deficient thymic lymphoma predisposition. This would occur in two different scenarios (Fig. 8). First, we consider TOP2-induced DSBs appearing independently of V(D)J recombination (Fig. 8, top). In this situation, a cd RAG2 RAG1 TOP2B RAD21 WT Atm –/– ENDseq 2 3 4 5 −5 kb 0 bp 5 kb WT Atm –/– Rag2 –/– WT Atm –/– Rag2 –/– ENDseq signal (RPKM) TOP2B-RAD21 peaks RAG peaks 4 6 8 10 −5 kb 0 bp 5 kb RAG1 RAG2 RAG1 RAG2 TOP2B IgG ChIPseq signal (RPKM) RAG (RAG1 + RAG2) 19,545 TOP2B 1003 42% b 13,289 1 −8 kb 0 bp 8 kb −8 kb 0 bp 8 kb −8 kb 0 bp 8 kb −8 kb 0 bp 8 kb −8 kb 0 bp 8 kb −8 kb 0 bp 8 kb 0 200 400 600 C2 C1 13,289 1 0 200 400 600 C2 C1 13,289 1 4 8 12 16 C2 C1 13,289 1 020 60 C2 C1 13,289 1 02468 C2 C1 13,289 1 02468 C2 C1 Fig. 6 TOP2B and cohesin colocalize with RAG genome-wide. a Heatmaps of RAG240, RAG140, TOP2B, RAD21 and ENDseq (DSBs) signal at RAG1 and RAG2 peaks (merged) in wild-type and Atm−/−primary thymocytes, as indicated. K-means clusters based on RAG1 and RAG2 signal are shown as C1 and C2. bOverlap of TOP2B and RAG peaks. Peaks of the RAG complex were defined as a merge of RAG1 and RAG2 proteins. cGlobal profile of TOP2B signal at RAG1 or RAG2 peaks. Control IgG signal is also shown. dGlobal profile of ENDseq signal at either RAG (merge of RAG1 and RAG2 peaks) or TOP2B- RAD21 (overlap between TOP2B and RAD21) peaks in wild-type, Atm−/−and Rag2−/−primary thymocytes. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14638-w ARTICLE NATURE COMMUNICATIONS | (2020) 11:910 | https://doi.org/10.1038/s41467-020-14638-w |www.nature.com/naturecommunications 9