PROJECT ABSTRACT/SUMMARY Aneuploidy and loss of heterozygosity are highly prevalent chromosome aberrations caused by sister- chromatid segregation errors during mitosis. Aneuploidy is especially common in triple-negative breast cancers, where outgrowth of aneuploid cells is permitted by the near-universal loss of TP53 that normally surveils genomic integrity. Prior work from our team has connected the aneuploid phenotype in breast cancer to network-level abundance changes in genes induced by mitotic transcription factors, which are chronically elevated in tumors. The target genes of these transcription factors reside upstream and downstream of the chromosome passenger complex (CPC), a four-protein sensor of proper spindle assembly and a mediator of repair. The CPC must localize to the inner centromere and auto-activate when microtubules are incorrectly attached. We recently found that the CPC accumulates during metaphase to a critical concentration causing it to phase separate as a liquid condensate. CPC phase separation likely confers robustness of function, yet cancers manage to bypass this checkpoint by changing the abundance of multiple regulators and effectors that together cause cells to enter a state of chromosome instability. Our objective is to unravel how the complex abundance imbalances of network regulators found in tumors stress the robust localization of the CPC to the inner centromere and generate chromosome instability in breast cancers or precursor lesions. The leading hypothesis is that phase-separated CPC acts as a “phenotypic capacitor” during mitosis by buffering small to moderate imbalances (storage) and unleashing dramatic rearrangements when a threshold imbalance is reached (discharge). We will test this hypothesis using biochemical reaction-diffusion models of spatially regulated CPC phase separation, which will be tailored to primary mammary organoids derived from a mosaic GEMM of triple-negative mammary cancer and extended to clinical samples through standard diagnostic assays. The specific aims are to 1) develop and validate a spatial systems model of CPC recruitment that isolates phase separation and predicts critical network imbalances in cancer-predisposed mammary organoids; 2) test the instability-generating potential of critical network imbalances by quantitatively perturbing triple- negative mammary premalignancies in vivo; and 3) leverage routine clinical diagnostics to predict druggable chromosomal instability signatures in any primary breast cancer. Patient-specific, systems-level models of aneuploidy susceptibility will nominate kinase inhibitors in the network that are predicted to shift cells from robust to fragile states of segregation fidelity.