TY  - GEN
TI  - Investigating the mechanistic basis of chromothripsis initiation and the clonal evolution of chromothriptic cells
AV  - public
ID  - heidok37382
CY  - Heidelberg
N2  - Chromothripsis is a form of genomic instability characterized by massive chromosomal shattering, resulting in complex rearrangements. This phenomenon affects 30-50% of all cancers and is strongly linked to poor prognosis. TP53 mutations are tightly associated with chromothripsis.
Although several mechanisms have been proposed, the specific triggers, molecular processes driving chromothripsis and the precise sequence of events are still unclear. In particular, how TP53 loss exactly drives replication stress and genomic instability, ultimately leading to such catastrophic events, is poorly understood. Furthermore, the timing of telomere stabilization, which is essential for chromothriptic cells to survive and evade apoptosis, remains unresolved. Moreover, our current knowledge of chromothripsis initiation is largely based on artificially induced in vitro systems or retrospective genomic analyses of fully developed tumours.
To address these challenges, I used skin-derived fibroblasts from Li-Fraumeni Syndrome (LFS) patients, which undergo spontaneous chromothripsis. In these fibroblasts, bulk whole genome sequencing (WGS) data showed chromothripsis at late passages (TP53?/?), whereas the matched early-passage (TP53+/?) cells did not show clonal chromothripsis. Through a combination of phenotypic assays and cutting-edge multiomics approaches, I performed a longitudinal study to investigate two major questions: i) what leads to chromothripsis, and ii) what gives chromothriptic cells a selective advantage to become dominant in a cell population.
The longitudinal profiling revealed that as early-passage cells advanced towards the growth crisis, they lost the wildtype TP53 allele, thus triggering global hypertranscription and increased nucleotide consumption. Simultaneously, dysfunctional p53 impaired the pentose phosphate pathway, leading to reduced nucleotide synthesis, as evidenced by transcriptomic and proteomic analyses. This imbalance resulted in replication stress induced by a competition between transcription and replication for the limited nucleotide pool. As a consequence of replication stress and checkpoint dysregulation due to p53 functional loss, LFS cells exhibited increased telomere attrition, breakage-fusion-bridge (BFB) cycles, chromatin bridges, multipolar spindle formation, chromosome missegregation, and micronuclei formation. This cascade of events ultimately led to chromothripsis and massive complex rearrangements. In addition, Single-cell DNA analysis revealed a high heterogeneity across cells, along with frequent copy number losses, rare events and diverse chromothriptic events already at early passages. Conversely, post-crisis chromothriptic cells exhibited clonal chromothripsis and complex rearrangements.
Notably, while most cells underwent apoptosis or negative selection, I found that a small subset of cells survived due to selective advantages and the activation of key adaptation mechanisms.
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These included telomere stabilization via alternative lengthening of telomeres (ALT) or TERT activation, extrachromosomal circular DNA (ecDNA) formation, gene fusions, oncogene activation, and loss of tumour suppressor genes. The activation of such mechanisms conferred the cells survival advantages, driving rapid expansion and clonal evolution. Thus, I captured the sequence of events, from the non-neoplastic cell population to the appearance of dominant clones carrying chromothriptic chromosomes.
Taken together, this study unravels the complex interplay between p53 dysfunction, nucleotide metabolism, and genomic instability in the initial phases of chromothripsis, thus providing crucial insights into the first steps of chromothripsis-mediated cancer development. Consequently, these findings may guide future strategies for cancer prevention and therapeutic interventions.
A1  - Philippos, George
Y1  - 2025///
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/37382/
ER  -