Wednesday, April 2, 2025
4/2/2025
A systems-level approach to therapeutically target STING in cancer - PROJECT SUMMARY Most proteins have more than one function and participate in a wide range of biological processes. This compli- cates our ability to infer protein function from sequence. This problem, otherwise known as genotype-to-pheno- type mapping, represents one of biology’s most intractable challenges. One way by which proteins diversify their functionality is through the evolution of modular domains, each of which can engage in distinct functions. Satu- ration mutagenesis, which systematically alters each amino acid in a given protein, is a powerful technique to map diverse functions to individual domains by generating separation-of-function mutants. However, the utility of this approach remains severely limited by excessive labor, prohibitive costs, and, most importantly, low-di- mensional readouts. The vast majority of saturation mutagenesis libraries have thus far been screened in vitro using biochemical or simple cellular assays. There are currently no approaches to probe the complex cellular and in vivo functions of saturation mutagenesis libraries at scale. This inspired our development of SatSeq – a modular breakthrough technology that couples the power of single cell sequencing with saturation mutagenesis and DNA barcoding – to map the structure-function landscape of multimodal proteins in complex and dynamic biological systems both in vivo and in vitro. As a proof of principle, we will develop SatSeq to interrogate the opposing functions of an immune-related protein called Stimulator of Interferon Genes (STING) in cancer pro- gression. STING is a master regulator of innate immunity, and it has been proposed to activate anti-tumor im- mune responses early in disease progression. Yet, recent evidence suggests that its chronic activation in cancer cells can drive metastasis. We hypothesize that this duality is achieved through distinct functional elements. Coupling genome-wide, single cell transcriptional profiling with in vivo interrogation of saturation mutagenesis barcodes, we will test the modular and time-dependent functions of STING during cancer progression. We will then utilize SatSeq as a target validation platform for STING modulators in clinical development, given its ability to readily identify resistance mutations that prevent drug-target binding without impacting target protein activity (i.e. gatekeeper mutations). The identification of such mutations is the gold standard for validating on-target drug effects, which will be applied here to a set of small molecule activators and inhibitors of STING. This is particularly salient since the first STING agonist to be used in clinical trials (DMXAA) was ineffective due to its inability to bind its target. In sum, we expect SatSeq will have significant impact at the fundamental and translational levels. First, it will expand our understanding of STING’s diverse functions during cancer progression – revealing the context in STING which activation vs. inhibition is therapeutically beneficial and creating new avenues for patient selection. Second, as a high-throughput platform to validate on-target drug activity, SatSeq will determine whether emerging STING modulators act through on-target effects and suggest mechanisms of drug action.
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