Novel target deconvolution in cGAS-STING pathway

免疫疗法代表着癌症治疗方面的最新突破，这是由于对转化细胞如何颠覆我们的免疫监控的加速机制的理解。因此，发展治疗性 以可控的方式加强系统免疫监控的策略已成为该领域的主要兴趣之一。在不同的药物中，cGAS-STING途径的小分子激动剂最近引起了人们的关注，因为它们有望与免疫治疗协同作用，通过上调干扰素应答来增强抗癌免疫反应。 我们之前发现了一种化合物BDW568，它被证明以一种刺痛依赖的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BDW568的直接靶点(S)。因此，观察到的表型表明，BDW568通过(1)与cGAS-STING通路的未知调节因子结合，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BDW568与先前发现的表型相关的细胞靶标。在第一种方法中，我们将合成基于BDW568的光亲和探针，并使用它们标记任何与BDW568结合的蛋白质。标记的目标(S)将通过全细胞裂解物下拉实验与质谱仪(MS)相结合来鉴定。在第二种方法中，我们将使用CRISPR-Cas9单独敲除所有已知与刺痛或与干扰素途径相关的基因。BDW568靶基因的敲除应表现出对该化合物的抵抗或基础干扰素水平的升高，而不存在BDW568通过刺痛。从这些方法中的任何一种出现的任何目标候选都将根据现场标准进行严格验证，以确定观察到的表型是由于目标参与。拟议研究的成功完成将扩大cGAS-STING途径的靶向空间。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天然免疫反应的理解。

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.