Wednesday, January 15, 2025
1/15/2025
PROJECT SUMMARY Chromosome missegregation or errors in cytokinesis produce aneuploidy, a chromosome content other than a multiple of the haploid number. The linkage of aneuploidy to tumorigenesis has long been recognized. A striking chromosomal abnormality linked to chromosome missegregation is chromothripsis (also known as chromoanagenesis), an event in which one (or two) chromosomes appear to have been shattered into tens to hundreds of small genomic fragments and religated back together in random order. Chromotriptic chromosomes are now recognized to be present in a broad range of cancers. With support from an NIGMS R35 grant, we have identified mechanisms of normal chromosome segregation that act to prevent aneuploidy in the normal situation and have determined that single chromosome missegregation or transient spindle pole amplification is a driver of tumorigenesis. We have identified the epigenetic mark of centromere identity and determined that DNA replication acts as an error correction mechanism to maintain that identity. We have identified key molecular mechanisms underlying the mitotic checkpoint (also known as the spindle assembly checkpoint), the primary guard against chromosome missegregation in mammals. We have identified how both mitotic checkpoint activation and silencing involve the catalytic action of a conformation altering AAA+ ATPase TRIP13. We have also determined that mitotic exit has an absolute requirement for TRIP13-mediated disassembly of the checkpoint inhibitor or the non-essential APC15 subunit of the E3 ubiquitin ligase that targets mitotic cyclin destruction. By exploiting a unique feature of the human Y centromere, we have produced cells in which we can induce selective, transient inactivation of the Y centromere, with the Y chromosome missegregated into micronuclei at high frequency. With these and whole genome sequencing, we determined that simple missegregation into a micronucleus can initiate chromothripsis and drive the complex genome rearrangements frequently found in human cancer. In the upcoming 5 years, we propose to determine mechanisms of fragmentation of a chromosome during chromothripsis, identify and validate nucleases that fragment micronuclear chromosomes, determine how shattered chromosomes are reassembled and produce extrachromosomal DNA (ecDNA), determine mechanisms of inheritance of ecDNA, and determine the role of spatial proximity in the inheritance of centromere identity, including neocentromere formation and other genomic abnormalities. We will also exploit our development over the last 15 years of antisense oligonucleotide (ASO) therapy for nervous system disease to undertake proof of principle therapy development targeting inactivation of the mitotic checkpoint by testing suppression of TRIP13/APC15 for the major brain cancer glioblastoma.
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