Recent developments in sequencing technologies led to the discovery of a novel form of genomic instability, termed chromothripsis. This catastrophic genomic event, involved in tumorigenesis, is characterized by tens to hundreds of simultaneously acquired locally clustered rearrangements on one chromosome. We hypothesized that leukemias developing in individuals with Ataxia Telangiectasia, who are born with two mutated copies of the ATM gene, an essential guardian of genome stability, would show a higher prevalence of chromothripsis due to the associated defect in DNA double-strand break repair. Using whole-genome sequencing, fluorescence in situ hybridization and RNA sequencing, we characterized the genomic landscape of Acute Lymphoblastic Leukemia (ALL) arising in patients with Ataxia Telangiectasia. We detected a high frequency of chromothriptic events in these tumors, specifically on acrocentric chromosomes, as compared with tumors from individuals with other types of DNA repair syndromes (27 cases total, 10 with Ataxia Telangiectasia). Our data suggest that the genomic landscape of Ataxia Telangiectasia ALL is clearly distinct from that of sporadic ALL. Mechanistically, short telomeres and compromised DNA damage response in cells of Ataxia Telangiectasia patients may be linked with frequent chromothripsis. Furthermore, we show that ATM loss is associated with increased chromothripsis prevalence in additional tumor entities.

Chromothripsis and chromoanasynthesis are catastrophic events leading to clustered genomic rearrangements. Whole-genome sequencing revealed frequent complex genomic rearrangements (n = 16/26) in brain tumors developing in mice deficient for factors involved in homologous-recombination-repair or non-homologous-end-joining. Catastrophic events were tightly linked to Myc/Mycn amplification, with increased DNA damage and inefficient apoptotic response already observable at early postnatal stages. Inhibition of repair processes and comparison of the mouse tumors with human medulloblastomas (n = 68) and glioblastomas (n = 32) identified chromothripsis as associated with MYC/MYCN gains and with DNA repair deficiencies, pointing towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements.

Chromothripsis and chromoanasynthesis are catastrophic events leading to clustered genomic rearrangements. Whole-genome sequencing revealed frequent chromothripsis or chromoanasynthesis (n= 16/26) in brain tumors developing in mice deficient for factors involved in homologous-recombination-repair or nonhomologous-end-joining. Catastrophic events were tightly linked to Myc/Mycn amplification, with increased DNA damage and inefficient apoptotic response already observable at early postnatal stages. Inhibition of repair processes and comparison of the mouse tumors with human medulloblastomas (n=68) and glioblastomas (n=32) identified chromothripsis as associated with MYC/MYCN gains and with DNA repair deficiencies, pointing towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/314518 doi: bioRxiv preprint first posted online May. 4, 2018; 3 Chromothripsis and chromoanasynthesis are two forms of genomic instability that lead to complex genomic rearrangements affecting one or very few chromosomes [1][2][3] . These two types of catastrophic events play a role in numerous tumor entities as well as in some congenital diseases 3,4 . The first form, chromothripsis, is characterized by the simultaneous occurrence of tens to hundreds of clustered DNA double-strand breaks 1,5 . The DNA fragments resulting from this shattering event are re-ligated by error-prone repair processes, with some of the fragments being lost. The outcome is a highly rearranged derivative chromosome, with oscillations between two or three copy-number states 6 . Conversely, the local rearrangements arising from chromoanasynthesis exhibit altered copy number due to serial microhomologymediated template switching during DNA replication 2 . Resynthesis of fragments from one chromatid and frequent insertions of short sequences between the rearrangement junctions are associated with copy number gains and retention of heterozygosity 2 .The availability of murine tumor models recapitulating these phenomena would substantially facilitate the investigation of the mechanistic aspects underlying chromothripsis and chromoanasynthesis. We showed previously the role of constitutive and somatic DNA repair defects in catastrophic genomic events in the context of TP53 and ATM mutations 5,7 . Further factors essential to the biochemical and signaling context of occurrence of these catastrophic events remain to be identified, and the role of repair processes in chromothripsis and chromoanasynthesis needs to be better defined.Homologous recombination repair (HR) and canonical non-homologous endjoining (cNHEJ) represent the two major repair processes for DNA double-strand breaks in mammalian cells. Conditional inactivation of key repair factors of either of these t...

Chromothripsis is a form of genome instability, whereby a presumably single catastrophic event generates extensive genomic rearrangements of one or few chromosome(s). However, little is known about the heterogeneity of chromothripsis across different clones from the same tumor, as well as changes in response to treatment. We analyzed single-cell genomic and transcriptomic alterations linked with chromothripsis in human p53-deficient medulloblastoma (n=7). We reconstructed the order of somatic events, identified early alterations likely linked to chromothripsis and depicted the contribution of chromothripsis to malignancy. We characterized subclonal variation of chromothripsis and its effects on double-minute chromosomes, cancer drivers and putatively druggable targets. Furthermore, we highlighted the causative role and the fitness consequences of specific rearrangements in neural progenitors.

In vitro assays for clustered DNA lesions will facilitate the analysis of the mechanisms underlying complex genome rearrangements such as chromothripsis, including the recruitment of repair factors to sites of DNA double‐strand breaks (DSBs). We present a novel method generating localized DNA DSBs using UV irradiation with photomasks. The size of the damage foci and the spacing between lesions are fully adjustable, making the assay suitable for different cell types and targeted areas. We validated this setup with genomically stable epithelial cells, normal fibroblasts, pluripotent stem cells, and patient‐derived primary cultures. Our method does not require a specialized device such as a laser, making it accessible to a broad range of users. Sensitization by 5‐bromo‐2‐deoxyuridine incorporation is not required, which enables analyzing the DNA damage response in post‐mitotic cells. Irradiated cells can be cultivated further, followed by time‐lapse imaging or used for downstream biochemical analyses, thanks to the high throughput of the system. Importantly, we showed genome rearrangements in the irradiated cells, providing a proof of principle for the induction of structural variants by localized DNA lesions.

In vitro assays for clustered DNA lesions will facilitate the analysis of the mechanisms underlying complex genome rearrangements such as chromothripsis, including the recruitment of repair factors to sites of DNA double-strand breaks. We present a novel method generating localized DNA double-strand breaks using UV-irradiation with photomasks. The size of the damage foci and the spacing between lesions are fully adjustable, making the assay suitable for different cell types and targeted areas. We validated this set-up with genomically stable epithelial cells, normal fibroblasts, pluripotent stem cells and patient-derived primary cultures. Our method does not require a specialized device such as a laser, making it accessible to a broad range of users. Sensitization by BrdU incorporation is not required, which enables analyzing the DNA damage response in post-mitotic cells. Irradiated cells can be cultivated further, followed by time-lapse imaging or used for downstream biochemical analyses, thanks to the high-throughput of the system. Importantly, we showed genome rearrangements in the irradiated cells, providing a proof of principle for the induction of structural variants by localized DNA lesions. * *** 0 J 1 0 0 J 2 0 0 J 3 0 0 J 0 1000 2000 3000 Energy applied γH2AX foci (mean intensity)

Recent developments in sequencing technologies lead to the discovery of a novel form of genome instability, termed chromothripsis. This catastrophic genomic event, involved in cancer formation, is characterized by tens to hundreds of locally clustered rearrangements on one chromosome, acquired simultaneously. We hypothesized that leukemias developing in individuals with Ataxia Telangiectasia, who are born with two mutated copies of the ATM gene, essential guardian of genome stability, would show a higher prevalence for chromothripsis due to the defect in DNA double-strand break repair. Using whole-genome sequencing, fluorescence in situ hybridization and RNA sequencing, we characterized the genomic landscape of Acute Lymphoblastic Leukemia (ALL) in patients with Ataxia Telangiectasia. We detected a high frequency of chromothriptic events in these tumors, specifically on acrocentric chromosomes, as compared to tumors from individuals with other types of DNA repair syndromes (27 cases in total, of which 10 with Ataxia Telangiectasia). Our data show that the genomic landscape of Ataxia Telangiectasia ALL is clearly distinct from that of sporadic ALL. Mechanistically, short telomeres and compromised DNA damage response in cells of Ataxia Telangiectasia patients are linked with frequent chromotripsis. Additionally, we show that ATM loss is associated with increased chromothripsis prevalence in further tumor entities. Citation Format: Manasi Ratnaparkhe, Mario Hlevnjak, Thorsten Kolb, Anna Jauch, Kendra Maass, Frauke Devens, Agata Rode, Volker Hovestadt, Andrey Korshunov, Agata Pastorczak, Wojciech Mlynarski, Stephanie Sungalee, Jan Korbel, Jessica Hoell, Ute Fischer, Till Milde, Christof Kramm, Michaela Nathrath, Krystyna Chrzanowska, Eugen Tausch, Masatoshi Takagi, Takashi Taga, Shlomi Constantini, Jan Loeffen, Jules Meijerink, Stefan Zielen, Gudrun Goehring, Brigitte Schlegelberger, Eberhard Maass, Reiner Siebert, Joachim Kunz, Andreas Kulozik, Barbara Worst, David Jones, Stefan Pfister, Marc Zapatka, Peter Lichter, Aurelie Ernst. Genomic profiling of acute lymphoblastic leukemia in ataxia telangiectasia patients reveals tight link between ATM mutations and chromothripsis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 509. doi:10.1158/1538-7445.AM2017-509

Chromothripsis and chromoanasynthesis are two forms of genomic instability leading to complex genomic rearrangements that affect one or very few chromosomes. These one-off catastrophic events play a role in numerous tumor entities as well as in some congenital diseases. The availability of murine models recapitulating both phenomena would substantially facilitate the investigation of the mechanistic aspects underlying catastrophic genomic events. Homologous recombination repair (HR) and canonical Non-Homologous-End-Joining (cNHEJ) represent the two major processes for DNA double-strand break repair in mammalian cells. Conditional inactivation of key factors of either of these two pathways, such as Brca2 for HR and Xrcc4 or Lig4 for cNHEJ in nestin-expressing or Emx1-expressing murine neural progenitor cells leads to medulloblastomas and gliomas in a p53-deficient background. We showed by whole-genome sequencing that these tumors frequently display chromothripsis or chromoanasynthesis (33 to 73% of the analyzed tumors, n= 27) and that catastrophic rearrangements drive tumor development. FISH analysis identified a link between chromoanasynthesis and increased numerical and structural aberrations and with the presence of marker chromosomes. In addition, amplifications of c-Myc and n-Myc likely facilitate catastrophic events. Detailed analysis of the microhomologies at the breakpoint junctions on the chromosomes affected by complex genomic rearrangements identified cNHEJ and alternative end-joining as likely repair processes involved in chromothripsis and chromoanasynthesis. Treatment of cells derived from the mouse tumors with inhibitors of HR and/or alternative end-joining (e.g. RAD51 and PARP inhibitors, respectively) in combination with DNA damage revealed the dependence of these tumor cells on specific repair processes and showed that these DNA repair deficiencies can be utilized for synthetic lethality approaches. Comparison of the mouse tumors with whole-genome sequencing data from human medulloblastomas (n=68) and gliomas (n=32) identified an association between chromothripsis and deficiencies in repair processes, by analyzing copy-number level aberrations affecting repair factors and mutational signatures of DNA double-strand break repair defects. This link between DNA repair deficiency and chromothripsis was further confirmed in additional tumor entities such as breast cancer (n=356) and melanoma (n=69). In analogy to the clinical use of PARP inhibitors in the context of BRCA-deficient breast cancer, our findings point towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements. Citation Format: Manasi Ratnaparkhe, John Wong, Pei-Chi Wei, Mario Hlevnjak, Paul Northcott, David T. Jones, Marcel Kool, Anna Jauch, Agata Pastorczak, Andrey Korshunov, Rajiv Kumar, Susanna M. Downing, Stefan M. Pfister, Marc Zapatka, Peter J. McKinnon, Frederick W. Alt, Peter Lichter, Aurelie Ernst. Inactivation of factors of DNA double-strand break repair by homologous recombination or non-homologous end-joining leads to frequent catastrophic genomic events in murine and human tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1352.