Dissecting chromothripsis, one step at a time

• Applicant: Privatdozentin Dr. Aurélie Ernst
• Subject Area: General Genetics and Functional Genome Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 460595631

Chromothripsis is a recently identified form of genome instability, by which a presumably single catastrophic event generates extensive genome rearrangements of one or a few chromosome(s). Widely considered as an early event in tumor development, this phenomenon plays a causative role in tumor onset. Chromothripsis is now recognized as frequent in cancer, occurring in more than 20% of cases in numerous tumor types. Importantly, chromothripsis is linked with poor prognosis. Even though chromothriptic events play a major role in tumor development, direct evidence of what predisposes cells to chromothripsis and what is the exact sequence of events in this process are lacking. Furthermore, it remains unclear how chromothriptic cells come to dominate tumor cell populations. We recently characterized a unique model system allowing to directly capture each step of the chromothriptic process. Primary cultures from patients with Li-Fraumeni syndrome (germline mutation in TP53) display no major rearrangement at early passages but show spontaneous chromothripsis occurrence at late passages. In Goal 1, we will identify what key alterations lead to chromothripsis, longitudinally map the clonal dynamics of how chromothriptic cells arise and come to dominate cell populations, and identify key transition points to mechanistically test. Starting from cultured patient-derived cells with no complex rearrangement and going up to spontaneous chromothripsis occurrence, we will dissect clonal evolution by single-cell genomics and phenotypic profiling. We will capture the process by longitudinal single-cell DNA sequencing and quantify over time mechanistic features linked with chromothripsis (e.g. telomere hallmarks, micronuclei formation, replication stress) to explain cellular processes associated with the appearance of dominant chromothriptic clones. In Goal 2, we will dissect the mechanistic role of SETD2 in chromothripsis. We identified the SETD2 methyltransferase as strongly linked with chromothripsis and SETD2 inactivation as an early event in medulloblastomas with chromothripsis. We will characterize the specific signal transduction and transcriptional regulatory machinery promoting chromothripsis downstream of SETD2. We will perform CRISPR gene editing followed by the analysis of complex genome rearrangements and phenotypic features linked with chromothripsis in the engineered lines. Beyond SETD2, we will analyze other critical factors that potentially control chromothripsis (identified in Goal 1) using functional genomics. Through a multi-disciplinary approach combining single-cell genomics, bioinformatics and functional studies, and using unique models that we established, we will decipher the genetic and cellular mechanisms giving rise to chromothripsis. A better understanding of the underlying biological processes leading to chromothripsis will be a prerequisite for laying the basis for the development of novel therapeutic strategies.

• DFG Programme: Research Grants