Probing cytosolic nucleic acid sensing pathways in cancer

摘要 哺乳动物细胞引发炎症的能力是许多过程的核心，包括胚胎发生， 伤口愈合、组织再生和癌症转移。炎症信号的主要来源是 细胞质中双链（ds）核酸的异常存在。哺乳动物细胞进化得很高- 检测胞质核酸作为抗病毒防御的保守机制。在正常细胞中，cGAS（cy- 环GMP-AMP合酶）及其下游信号传导效应物STING（干扰素基因刺激物）具有 被认为是I型干扰素（IFN）信号传导和下游免疫激活的必需介质。 然而，我们已经表明，在染色体不稳定性（CIN）的癌细胞中，没有I型的证据。 尽管存在胞质DNA和cGAS和STING的组成型激活，但IFN信号传导仍然存在。相反地， 癌细胞在STING的下游重新连接它们的信号传导，以选择性地抑制IFN信号传导，并使其他细胞能够 er促转移途径如NF-κB。三个重要的证据提出了质疑的埃森- cGAS-STING途径在促进抗肿瘤免疫中的有效性，并提示迄今未被认识到的 癌症中核酸传感的冗余和背景依赖性：1）染色体不稳定的癌症 细胞保持对胞质dsRNA的IFN-应答性。2)CIN的癌细胞仍然可以引发一种强大的抗肿瘤 以不依赖于cGAS-STING和I型IFN的方式对胞质dsDNA的免疫应答。3)表达 核酸传感器和下游炎症通路的变化在肿瘤亚群中高度可变 和转移性细胞状态-其中出现干细胞样至更定型的上皮祖细胞的连续体， 服务。总之，这些发现挑战了目前的观点，即cGAS-STING信号是通用的媒体- 对胞质dsDNA应答的炎症反应。在这里，我们的目标是了解功能冗余， 跨胞质核酸传感途径的相互作用以及它们的转录输出如何随时间变化。 莫尔细胞分化状态。我们将系统地询问关键的核酸传感器及其下游 在三种同基因小鼠模型中的效应子，其特征在于增加的转移潜力和高水平的 CIN。我们将实验性地操纵CIN率以鉴定细胞溶质核酸依赖性，但cGAS- 免疫激活的非STING依赖性机制（目的1）。然后我们将高通量单细胞 用组合CRISPR介导的关键胞质核酸传感器的基因失活进行测序， 通过SOX 2表达区分的转移起始干细胞中的效应物，与其更分化的干细胞相比， 对应物，以映射该途径的细胞状态特异性调控逻辑（Aim 2）。把背景弄清楚- 依赖这种极其重要和通用的信号级联有可能改变我们的生活。 思考癌症中的慢性炎症，并揭示染色体不稳定的治疗弱点， 这些癌细胞在其他方面对cGAS-STING信号传导具有抗性。

ABSTRACT 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-κB. 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.