Abstract 4126: Illuminating the dark kinome using proximity labeling and phosphoproteomics analysis

Kinases are highly druggable targets for cancer therapy due to their control over a variety of oncogenic processes via protein phosphorylation. While, large clinical phosphoproteomics datasets have expanded our understanding of how the phosphoproteome is altered in cancer, annotation of the phosphoproteome is significantly lacking. 95% of phosphorylation sites in the PhosphoSitePlus database have no associated kinase, and 80% of annotated substrates are assigned to just 20% of the most well-studied kinases. Of the ∼650 kinases in the human genome, 162 are designated by the NIH as ‘dark kinases’ due to lack of information on their function and substrates. The primary aim of my research is to define the functions and substrates of the dark kinome and identify kinases with therapeutic potential.My lab has collected mass spectrometry (MS)-based proximity labeling data for 137 of the dark kinases. Probabilistic scoring of raw data yields functionally relevant proximity networks for each kinase that defines its cellular localization and protein complex membership. Enrichment analysis identifies functional protein domains and protein sequence motifs that drive kinase localization and substrate specificity via protein-protein interactions. Additionally, clustering of this data highlights the prevalence of kinase signaling hubs – distinct locations within the cell harboring diverse kinase populations – that regulate extracellular signal processing, protein trafficking, transcription, and translation.As a second branch of my work, I have developed kinase gain-of-function (GOF) phosphoproteomics experiments to identify substrates and pathways regulated by dark kinases. This medium-throughput method uses multiplexed deep phosphoproteomic profiling to measure changes in protein phosphorylation levels following transient overexpression of a kinase. Analysis of this data reveals signaling pathways regulated by a kinase, which can be validated using low-throughput western blots. When combined with proximity data, I am able to define high-confidence putative substrates that are both proximal to the kinase and significantly hyperphosphorylated following overexpression of the kinase. As an example of this workflow, I highlight the dark kinase BRSK2. In my BRSK2 GOF experiment, I identify 584 candidate sites that are significantly upregulated. Enrichment analysis of these sites reveals that BRSK2 is a positive regulator of AMPK and negative regulator of mTOR signaling. This is validated by western blotting of key AMPK and mTOR substrates. Mapping upregulated sites onto the proximity network for BRSK2 yields a small list of 39 high-confidence proximal substrates, an improvement on the 4 substrates in the PhosphoSitePlus database. Citation Format: Isaac Plutzer, Dhaval P. Bhatt, Emily M. Wilkerson, Regan Williamson, Dennis Goldfarb, M. Ben Major. Illuminating the dark kinome using proximity labeling and phosphoproteomics analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4126.