PROJECT SUMMARY The goal of this proposal is to discover how ongoing chromosome missegregation events in cancer cells (a process called chromosomal instability, or CIN) alters the tumor ecosystem to promote cancer progression. Chromosome copy number alterations (also referred to as aneuploidy) have long been associated with immune suppressive phenotypes, drug resistance, and metastasis. Beyond aneuploidy, however, it remains unknown whether the ongoing process of chromosome missegregation gives rise to tumor progression. Harnessing ex- perimental tools that enable us to dial-up or dial-down chromosome missegregation rates in otherwise isogenic backgrounds, we have previously found that ongoing chromosome missegregation generates rupture-prone mi- cronuclei, which expose genomic double-stranded DNA (dsDNA) to the cytosol, leading to persistent activation of the cGAS-STING innate immune pathway (1). Yet, instead of promoting a robust type I interferon (IFN) re- sponse, STING activation in cancer cells with CIN promotes noncanonical NF-kB (nc-NF-kB) signaling – through an unknown mechanism – to drive metastasis. The extent to which CIN-driven metastasis is dependent on the immune system remains unknown. We made the surprising observation that CIN drives tumor progression in a cancer cell non-autonomous manner by shaping the interaction between cancer cells and the tumor microenvi- ronment (TME). This finding motivated the development of a fundamentally new, systems-level approach to evaluate the nature and conditional-dependence of cell-cell interactions in the TME called ContactTracing. This method exploits intrinsic biological variance captured by single cell RNA sequencing technologies, to infer cellu- lar responses to ligand-receptor mediated interactions without prior knowledge of downstream target genes. Combining this innovative computational tool with genetic perturbation of CIN and STING we found that CIN engenders a pro-metastatic TME by inducing a cancer cell-intrinsic ER-stress response. In Aim 1, we propose to mechanistically dissect the epistatic relationship between CIN, STING, and ER-stress in the progression of triple negative breast cancer (TNBC) to determine whether ER-stress can represent a therapeutic target in chro- mosomally unstable tumors. We will also test whether an ER-stress response underlies nc-NF-kB activation. Under Aim 2, we will improve causal inference of tumor-derived ligand effects and explore their molecular basis using gene regulatory networks to ask whether CIN-dependent cell-cell interaction networks are conserved across cancer types using both human data and mouse models of breast, pancreatic, and lung cancers. The amalgamation of these approaches, combined with our deep understanding of CIN in cancer, is poised to eluci- date the complex roles of CIN-induced STING signaling on tumor-immune crosstalk during disease progression. Importantly, this work is poised to reveal novel strategies aimed at targeting ch...