Novel target deconvolution in cGAS-STING pathway

免疫治疗代表了癌症治疗的最新突破，这是由于对转化细胞如何破坏我们的免疫监视的加速机制理解。因此，开发治疗 以可控方式增强系统免疫监视的策略已成为该领域的主要兴趣之一。在不同的试剂中，cGAS-STING途径的小分子活化剂（激动剂）最近引起了关注，因为它们预期与免疫疗法协同作用并通过上调干扰素应答来增强抗癌免疫应答。 我们先前鉴定了一种化合物BDW 568，该化合物显示以STING依赖性方式激活干扰素途径。令人惊讶的是，后续研究表明cGAS和STING不是BDW 568的直接靶标。因此，观察到的表型表明，BDW 568通过（1）结合cGAS-STING途径的未知调节剂，或（2）通过产生不依赖于内源性STING激动剂2 '，3'-cGAMP的未知信号传导分子发挥作用。在拟议的研究中，我们将使用两种正交策略确定BDW 568的细胞靶点，该靶点负责先前发现的表型。在第一种方法中，我们将合成基于BDW 568的光亲和探针，并使用它们来标记任何与BDW 568结合的蛋白质。将通过全细胞裂解物下拉实验结合质谱法（MS）鉴定标记的靶标。在第二种方法中，我们将使用CRISPR-Cas9单独敲除已知与STING相互作用或与干扰素途径相关的所有基因。BDW 568靶标的敲除应证明对化合物的抗性或通过STING在没有BDW 568的情况下基础干扰素水平的升高。从这些方法中的任一种出现的任何靶候选物将根据该领域的标准进行严格验证，以确定观察到的表型是由于靶向接合。成功完成拟议的研究将扩大cGAS-STING途径内的靶空间。这些见解将为开发佐剂免疫疗法带来更多机会，并扩大我们对哺乳动物细胞先天免疫反应的理解。

Immunotherapy represents a recent breakthrough in cancer treatment fueled by the accelerating mechanistic understanding of how transformed cells subvert our immunosurveillance. Therefore, developing therapeutic strategies that enhance systemic immunosurveillance in a controllable manner has become one of the major interests in this field. Among different agents, small molecule activators (agonists) of the cGAS-STING pathway have recently attracted attention because they are expected to synergize with immunotherapies and enhance anti-cancer immune response by upregulating the interferon response. We previously identified a compound, BDW568, that was shown to activate the interferon pathway in a STING-dependent manner. Surprisingly, follow-up studies demonstrated that cGAS and STING are not direct target(s) of BDW568. Therefore, observed phenotype suggests that BDW568 acts either through (1) binding to an unknown regulator of the cGAS-STING pathway, or (2) by generating an unknown signaling molecule independent of 2’,3’-cGAMP, the endogenous STING agonist. In the proposed study we will identify the cellular target of BDW568 responsible for the previously discovered phenotype using two orthogonal strategies. In the first approach, we will synthesize BDW568-based photoaffinity probes and use them to label any protein that binds to BDW568. Labeled target(s) will be identified by a whole-cell lysate pulldown experiments coupled with mass spectrometry (MS). In the second approach, we will use CRISPR-Cas9 to individually knock out all the genes that are known to interact with STING or associate with interferon pathways. The knockout of the BDW568 target should demonstrate either resistance to the compound or elevation of the basal interferon level without BDW568 through STING. Any target candidate that emerges from either one of these approaches will be rigorously validated according to the standards of the field to establish that observed phenotype is due to on-target engagement. Successful completion of proposed studies will expand the target space within cGAS-STING pathway. These insights will lead to additional opportunities for developing adjuvant immunotherapies as well as expand our understanding of innate immune response in mammalian cells.