PROJECT SUMMARY Chromosomal instability (CIN) is a hallmark of cancer characterized by high rates of chromosome mis-segre- gation during cell division. CIN can generate nuclear aberrations termed micronuclei when a chromosome or chromosome fragment lags during anaphase and fails to join the main chromatin mass that will form the prima- ry nucleus. Micronuclei recruit nuclear envelopes but defects in construction lead to frequent rupturing, loss of compartmentalization, and an unregulated exchange of proteins and small vesicles with the cytoplasm. Mi- cronuclear envelope rupturing causes broad dysfunction and is associated with extensive DNA damage and genomic rearrangements, including clustered mutational phenomena such as chromothripsis and kataegis, which are commonly observed in cancer genomes. Ruptured micronuclei can also activate the pro-inflammato- ry cGAS-STING pathway, which plays essential roles in anti-tumor immunity. These observations suggest that micronuclei may represent key platforms for genome evolution and immune activation in cancer. The mecha- nisms driving DNA damage and immune activation at micronuclei are poorly understood. The laboratory dis- covered that the endoplasmic reticulum (ER)-associated exonuclease TREX1, which is mutated in a variety of human immune diseases including Aicardi-Goutières Syndrome, accumulates at micronuclei upon micronu- clear envelope rupture where it resects micronuclear DNA and limits cGAS-STING activation. Therefore, TREX1 occupies central positions in key pathways with diverse roles in human health and disease. Conse- quently, there is strong rationale to understand mechanisms of TREX1 activity and engagement with cytosolic DNA. The long-term goals of the laboratory are to determine mechanisms of DNA damage, clustered mutage- nesis, and immune activation at sites of nuclear envelope rupture. The specific Aims of this proposal are to 1) Elucidate mechanisms of TREX1 structure and function, 2) Determine how TREX1 is recruited to micronuclei, and 3) Dissect pathways of micronuclear DNA damage. Each objective is supported by extensive preliminary data. Aim 1 will focus on a previously uncharacterized region in TREX1, which is essential for its ability to de- grade cytosolic DNA and inhibit cGAS activation. Aim 2 will build on results showing that TREX1 DNA binding function is dispensable for its localization to micronuclei, while its association with the ER is essential. Aim 3 will use a new method to purify micronuclei to dissect sources of micronuclear DNA damage. Taken together, these data will provide fundamental insights into cancer genome evolution, explain how previously uncharac- terized TREX1 mutations cause Aicardi-Goutières syndrome, and may identify new strategies to improve anti- tumor immunity.

PROJECT NARRATIVE Micronuclei are aberrant nuclear compartments that can form as a result of chromosome mis-segregation. Frequent loss of micronuclear envelope integrity exposes DNA to the cytoplasm, leading to genomic DNA re- arrangements and immune activation. Building on our prior work, we now propose to determine the mecha- nisms of DNA damage and immune activation at micronuclei.