The ability of mammalian cells to elicit inflammation is central to many processes including embryogenesis, wound healing, tissue regeneration, and cancer metastasis. A major source of inflammatory signaling is the aberrant presence of double-stranded (ds) nucleic acids in the cytoplasm. Mammalian cells have evolved high- ly conserved mechanisms to detect cytosolic nucleic acids as an anti-viral defense. In normal cells, cGAS (cy- clic GMP-AMP synthase) and its downstream signaling effector STING (stimulator of interferon genes) have been proposed as essential mediators of type I interferon (IFN) signaling and downstream immune activation. We have shown however, that in cancer cells with chromosomal instability (CIN), there is no evidence of type I IFN signaling despite the presence of cytosolic DNA and constitutive activation of cGAS and STING. Instead, cancer cells rewire their signaling downstream of STING to selectively suppress IFN signaling and enable oth- er pro-metastatic pathways such as NF-?B. Three important pieces of evidence bring into question the essen- tiality of the cGAS-STING pathway in promoting anti-tumor immunity and suggest heretofore unappreciated redundancies and context dependence of nucleic acid sensing in cancer: 1) chromosomally unstable cancer cells retain IFN-responsiveness to cytosolic dsRNA. 2) Cancer cells with CIN can still elicit a robust, anti-tumor immune response to cytosolic dsDNA, in a manner independent of cGAS-STING and type I IFN. 3) Expression of nucleic acid sensors and downstream inflammatory pathways is highly variable across tumor subpopulations and metastatic cell states ? in which a continuum of stem-like to more committed epithelial progenitors is ob- served. Together, these findings challenge the current view that cGAS-STING signaling is the universal media- tor of inflammation in response to cytosolic dsDNA. Herein, we aim to understand functional redundancies and interactions across cytosolic nucleic acid sensing pathways and how their transcriptional outputs vary with tu- mor cell differentiation status. We will systematically interrogate key nucleic acid sensors and their downstream effectors in three syngeneic mouse models characterized by increased metastatic potential and high levels of CIN. We will experimentally manipulate CIN rates to identify cytosolic nucleic acid-dependent, but cGAS- STING-independent mechanisms of immune activation (Aim 1). We will then couple high-throughput single-cell sequencing with combinatorial CRISPR-mediated gene inactivation of key cytosolic nucleic acid sensors and effectors in metastasis-initiating stem cells distinguished by SOX2 expression, versus their more differentiated counterparts, to map the cell state-specific regulatory logic of this pathway (Aim 2). Unraveling the context- dependence of this extremely important and versatile signaling cascade has the potential to transform our thinking about chronic inflammation in cancer and to reveal therapeutic vulnerabilities in chromosomally unsta- ble cancer cells that are otherwise resistant to cGAS-STING signaling.
The aberrant presence of double-stranded nucleic acids in the cytoplasm promotes a robust inflammatory re- sponse, which has galvanized interest in developing therapies that can sensitize cancer cells to the immune system. However, we found that cancer cells with chronic inflammation - fueled by genomic instability ? have adapted resistance to a key innate immune pathway called cGAS-STING, redirecting a normally lethal output towards alternative pathways that promote metastasis. Motivated by the discovery that these resistant cells can still be sensitized to immune attack in a manner independent of cGAS-STING, we aim to unravel the con- text-dependence of this versatile signaling cascade to test if restoring normal responses to inflammation can be used as an effective therapeutic strategy to sensitize tumors to immune therapy.
