Integrated mass spectrometry-based chemoproteomic and genomic technologies for studying dynamic kinase interactomes

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PROGRAM ABSTRACT: Integrated mass spectrometry-based chemoproteomic and genomic technolo- gies for studying dynamic kinase interactomes Dynamic changes in protein-protein and protein-DNA interactions (PPIs and PDIs) control most cellular pro- cesses, including cell signaling and transcription; devastating diseases rewire PPI and PDI networks to drive disease progression, therapy resistance, and immune escape. Novel methods for mapping dynamic interaction networks are, therefore, urgently required to identify disease mechanisms and drug targets. Protein kinases are critical regulatory nodes in most cellular PPI and PDI networks, are often dysregulated in disease, and are highly druggable with synthetic, ATP-competitive inhibitors. Accordingly. insights into how diseases utilize kinases to rewire PPI and PDI networks are extremely relevant for combating many diseases. Advances in quantitative mass spectrometry (MS) have revolutionized proteomics, yet, facile methods for the systematic, sensitive, and high-throughput profiling of kinase PPIs, locus-specific PDIs, and their dynamics are lacking. We will develop transformative methods that combine cell-permeable affinity probes with chemical crosslinking and proximity labeling to globally encode kinase interactomes in situ, followed by integrated LC-MS and sequencing analyses. Cellular plasticity drives physiological and pathological de- and transdifferentiation, and lineage switching, critically contributing to development, tissue repair, cancer metastasis, organ fibrosis, and therapy and immune escape in numerous diseases. To identify drug targets for combating these disease phenotypes, we pressingly need to understand the signaling and transcriptional network that underly cellular plasticity. Our studies of patho- logical kinome rewiring linked ~20% of human kinases to cellular plasticity, among them numerous understudied kinases. We found that 70% these kinases localize to the nucleus and interact with transcription factors and chromatin remodelers. We also found that cellular plasticity dynamically alters the post-translational modifica- tions (PTMs) and PPIs of these kinases. How plasticity pathways coordinate dynamic changes in PTM, PPI and PDI networks to systematically alter chromatin states and transcription, however, remains largely unknown, leav- ing critical molecular mechanisms and drug targets unexplored. We will develop streamlined workflows for stud- ying nuclear kinase dynamics, combining kinobead/LC-MS kinome profiling with global proteomics, epigenomics, and transcriptomics analyses, and our novel interactomic platforms, and apply these workflows to unravel how plasticity pathways spatiotemporally control kinases during cellular de- and transdifferentiation. To summarize, our program seeks to develop novel bioanalytical methods and workflows to systematically study dynamic kinase interactomes, and to illuminate the mechanisms of pathological cellular plasticity. Pursuing our goals, we created an ambitious, rigorous, and productive research program that fosters inclusiveness and creativity, training the next generation of scientific leaders in proteomics, cell signaling, and chemical biology.