Project Summary A nucleus-to-mitochondria nucleic acid-sensing pathway prevents bypass of age-associated proliferative boundaries Development of immortality as function of age is dependent on the ability of cells to escape from at least two distinct proliferative barriers, replicative senescence and crisis. Both serve as critical tumor-suppressors, but the pathways governing them are distinct. Replicative senescence is triggered by short functional telomeres, dependent on the p53/pRB tumor suppressor pathways and characterized by permanent cell cycle arrest and continued metabolism. When p53/pRB pathways are dysfunctional, senescence entry is compromised, and cells continue to proliferate until their telomeres become dysfunctional and chromosome fusions arise. This triggers replicative crisis, a p53/pRB-independent state, where the vast majority of cells rapidly succumb to cell death. However, rare cells can even overcome this barrier and become neoplastic, pointing to replicative crisis as one of the final barriers against age-associated tumor cell initiation. Recently, it was discovered that cell death in crisis is governed by macroautophagy through a pathway in which cytoplasmic DNA species from fused and broken chromosomes activate the cGAS-STING cytoplasmic DNA-sensing response that normally detects viral DNA. Suppression of autophagy allowed cells to bypass crisis and continue to proliferate, while accumulating genome instability. This discovery represented the first crisis-bypass system, which allowed the design of a CRISPR suppression screen aimed at identifying factors required to protect cells against age-associated cancer initiation. Another nucleic acid sensor, ZBP1 emerged as critical for the crisis program, which was confirmed by ZBP1 suppression allowing cells to proliferate beyond crisis. Here, in three synergistic aims it is proposed to decipher the mechanism underlying the ZBP1-dependent inhibition of cancer initiation. AIM1 will determine the interactions between dysfunctional telomeres, telomeric (TERRA) transcripts and ZBP1 and define the mechanisms of ZBP1-mediated innate immune signaling on mitochondria during crisis. AIM2 is designed to investigate the mechanism of the mitochondrial localization of the crisis-specific isoform of ZBP1 and its relevant interacting partners. Finally, the ability to allow cells to proliferate beyond crisis revealed the existence of a third previously unknown proliferative barrier against cancer initiation (called M3), which will be extensively characterized in AIM 3. Successful completion of these aims will shed new light on crosstalk between telomeres, mitochondria and inflammation (three established hallmarks of aging), the role of a telomere-to-mitochondria innate immune signaling pathway in the prevention of age-associated cancer and establish biomarkers and new approaches to understand the relevance of the new M3 proliferative barrier as tumor-suppressor.