PROJECT SUMMARY/ABSTRACT Genomic instability is a hallmark of cancer and can drive high rates of chromosome segregation errors during mitotic cell division, which can generate abnormal structures called micronuclei that entrap mis-segregated chromosomes. Micronuclei are susceptible to massive DNA damage, triggering the catastrophic pulverization of the entrapped chromosome into small DNA fragments, a process termed chromothripsis. In addition to generating genomic rearrangements that drive cancer development, DNA fragments from micronuclei can also mis-accumulate and persist in the cytoplasm. These cytoplasmic DNAs are detected by the cytosolic DNA sensor cGAS, resulting in the cell-autonomous activation of the STING pathway to trigger an innate immune response. Although the cGAS-STING signaling mechanisms are well defined, the fate of cytoplasmic DNAs following recognition by cGAS remains a critical knowledge gap. Here I propose to investigate non-cell autonomous roles of cytoplasmic DNAs, which hold critical implications in how genomically unstable cancer cells can elicit inter- cellular responses with the tumor microenvironment. I hypothesize that cytoplasmic DNAs derived from fragmented chromosomes in micronuclei become exported for uptake by neighboring cells. To test this hypothesis, I will leverage an experimental system enabling chromosome-specific induction of micronuclei and cytoplasmic DNA followed by tracking of specific cytoplasmic DNA fragments that harbor a selectable marker. In Aim 1, I will determine whether and how cytoplasmic DNAs are released into the extracellular environment to facilitate non-cell autonomous activation of the cGAS-STING pathway in adjacent cells. I will further determine whether extracellular DNA derived from the cytoplasm of host cells can be taken up by recipient cells, which will be monitored by live-cell imaging using a dCas9-based cytoplasmic DNA reporter. In Aim 2, I will track the incorporation of cytoplasmic DNAs into recipient cell nuclei and determine whether these fragments can integrate into the host genome. This will be studied using a combination of cytogenetics and whole-genome sequencing to investigate the possibility of lateral DNA transfer between human cells. Despite occurring frequently in bacteria, inter-cellular DNA transfer has been a longstanding challenge to test in the context of human cancer. These studies have potential for broad impact by advancing our understanding of cancer cell interactions, including the transfer of oncogenes and/or mutations from chromosomally unstable cancer cells to non-cancer cells in the tumor microenvironment.