Wednesday, April 2, 2025
4/2/2025
Mechanisms of Chromosome Shattering from Defective DNA Replication - PROJECT SUMMARY/ABSTRACT Mis-segregated chromosomes entrapped in micronuclei are susceptible to catastrophic shattering through a process termed chromothripsis. The resulting DNA fragments subsequently undergo error-prone re-ligation, in turn generating complex genomic rearrangements that are detected in approximately one-third of diverse tumor types. Chromothripsis drives the punctuated evolution of cancer genomes by rapidly rearranging individual chromosomes within a few cell cycles, which can simultaneously inactivate multiple tumor suppressor genes, generate oncogenic gene fusions, and/or amplify cancer-associated genes as circular extrachromosomal DNA elements. Therefore, a complete mechanistic understanding of the etiology of chromothripsis represents a critical need toward defining how cancer cells acquire complex genomic alterations. Although chromothripsis may be partially triggered by DNA damage within micronuclei during interphase, recent evidence support a second wave of extensive DNA damage that is inflicted upon mitotic entry. The mechanisms underlying such mitotic DNA damage and its contributions to chromothripsis are poorly understood. To address this knowledge gap, we leveraged a chromosome-specific micronucleus and chromothripsis platform to conduct pooled CRISPR/Cas9 screens targeting the mammalian DNA damage response. We unexpectedly identified multiple components of the Fanconi anemia (FA) pathway as a requirement for chromothripsis. Although the FA pathway is normally a genome-protective DNA repair mechanism, our preliminary studies indicate that it functions abnormally during mitotic entry to induce extensive fragmentation of under-replicated chromosomes in micronuclei followed by mitotic DNA synthesis. We hypothesize that pathological activation of the FA pathway triggers chromothripsis during mitosis through the cleavage of incomplete DNA replication intermediates that accumulate within micronuclei. To test this central hypothesis, we propose three complementary aims to define the molecular mechanisms driving the mitotic shattering of micronucleated chromosomes. First, we will comprehensively characterize intrinsic defects associated with micronuclear DNA replication and determine the cell cycle stages during which they arise (Aim 1). Next, we will investigate how the FA pathway functions to shatter micronucleated chromosomes during mitosis, including identifying the structure-specific nuclease(s) involved in inducing widespread mitotic chromosome cleavage (Aim 2). Lastly, we will examine the role of the FA pathway in generating complex genomic rearrangements and extrachromosomal DNAs in experimental models of chromothripsis and in FA patient cancer genomes (Aim 3). Successful completion of these aims will provide a detailed mechanistic understanding of how mitotic errors instigate rapid karyotypic changes in cancer genomes. These studies seek to provide paradigm-shifting insight into how defective DNA replication activates an uncharacterized function of the FA pathway in triggering chromothripsis and cancer genome instability.
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