Proteome-wide base editor screens to assess phosphorylation site functionality in immunosenescence

Abstract We often depict signaling pathways by a dozen or so landmark phosphorylation events that culminate in the transcription of a gene or induction of a phenotype. In reality, a cascade of hundreds to thousands of phosphorylation sites create dynamic biochemical networks that orchestrate essentially every cellular process

from expression of transcriptional programs and cell proliferation to migration and cytotoxic effector functions. T cell signaling is an important example. Signal transduction through the T cell receptor and co-stimulatory molecules is incredibly complex and leads to important but distinct downstream effects necessary for proper

immune responses to pathogens, cancer, and vaccines. As humans age, T cell signaling becomes detrimentally altered, leading to “immunosenescence” and less efficient protection from malignancies. Mass spectrometry has enabled the profiling of tens of thousands of phosphorylation sites from a diverse range of cells and tissues.

Unfortunately, functionally characterizing the tens of thousands of post-translational modifications cells use to coordinate essentially all cellular and organismal processes remains a fundamental challenge in biology. Here, we propose an innovative technology that will enable the functional assessment of thousands of phosphorylation

sites in high throughput. We will employ CRISPR-mediated base editors, which mutate codons in genomic DNA, coupled to phenotypic screens to probe the signaling events that lead to specific T cell stimulation-specific gene expression programs or proliferation responses. Preliminary data from our laboratory strongly suggests that we

can functionally screen tens of thousands of phosphorylation sites for their contribution to NFAT or NFKB signaling in stimulated T cells. In this proposal, we will further optimize this technology to create a robust, reliable screening platform. To validate our screening technology, we will characterize a subset of

phosphorylation site mutants using conventional genomic engineering methods to understand how our screening platform compares to classic approaches to study phosphorylation site function, i.e. one mutation at a time. We will also extend this approach to primary human CD8+ T cells, to show that we can uncouple the signaling events

that lead to cell proliferation or expression of the activation/exhaustion marker PD1. High throughput functional screening of phosphorylation sites in primary immune cells will revolutionize the way we study signaling pathways and cellular decision making, and the way we approach drug development or adoptive cell therapies

to treat cancer and a variety of human ailments associated with aging.