Chromothripsis: unraveling the tumor evolution in vivo, from initiating tumor cells to therapy resistance

• Applicant: Privatdozentin Dr. Aurélie Ernst
• Subject Area: General Genetics and Functional Genome Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 512935482

Chromothripsis is a form of genome instability, by which a single catastrophic event generates extensive genome rearrangements of one chromosome. Generally considered as an early event in tumor development, this phenomenon is now recognized as frequent in cancer and linked with poor prognosis. However, direct evidence of initiating copy-number alterations occurring in vivo and how genetic clones become selected are lacking. Furthermore, the potential use of chromothripsis detection in liquid biopsies and how this may contribute to developing early detection tools or guiding treatment decisions has not been explored.First we will characterize the preneoplastic landscape of chromothriptic tumors in vivo. Nearly all patients with Li-Fraumeni syndrome (TP53 germline mutations) develop cancer, and these tumors show a close to 100% chromothripsis prevalence. We will dissect early-stage genomic alterations preceding tumor diagnosis in available blood cells from Li-Fraumeni syndrome individuals enrolled in surveillance programs (n=25). We will do single-cell DNA and RNA sequencing i) to identify clones with initiating copy-number alterations and cell types prone to such early copy-number alterations ii) to integrate early events with the genomic landscape of advanced stage cancers. Second we will use functional genomics to dissect the molecular effects of factors identified in Goal 1 and known candidate genes that potentially promote chromothripsis or lead to clonal expansion in chromothriptic tumors. These “factors” represent the specific signal transduction and transcriptional regulatory machinery leading to the development of chromothriptic tumors. We will perform a CRISPR-pooled screen of 15 candidate genes identified as putative promoters of clonal expansion, followed by the analysis of phenotypic features in the engineered lines (e.g. proliferation, apoptosis). In addition to candidates from Goal 1, we will dissect the mechanistic role of key players in the p53 pathway in chromothripsis. Third we will benchmark the use of chromothripsis detection in liquid biopsies. As part of the preliminary data for this proposal, we showed chromothriptic rearrangements in cell-free DNA from cancer patients. We will analyze the evolution of chromothriptic patterns in longitudinal liquid biopsies and identify fusion genes generated by chromothripsis by whole-genome and RNA sequencing of cell-free DNA, tumor tissue and matched germline controls (n=20 cases per tumor type, all samples are available). Finally, we will characterize extrachromosomal circular DNAs in tumors and matched cell-free DNA to get insights into the role of these structures in oncogene activation.By a multi-disciplinary approach using single-cell genomics, bioinformatics and functional studies, we will obtain a better understanding of the biological processes leading to chromothripsis, as a prerequisite for laying the basis for the development of novel surveillance and treatment strategies.

• DFG Programme: Research Grants