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Covalent Targeting of Splicing in T Cells

ABSTRACT: Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide chemical probe EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and availability of functional mRNA pools that can be depleted due to extensive alternative splicing. We further introduce a comprehensive list of proteins involved in splicing and leverage both cysteine- and protein-directed activity-based protein profiling (ABPP) data with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in primary human T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.

ORGANISM(S): Homo sapiens

PROVIDER: GSE279165 | GEO | 2024/10/09

REPOSITORIES: GEO

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Covalent Targeting of Splicing in T Cells.

Project description:Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and extensive ITK mRNA alternative splicing. We further introduce the most comprehensive list to date of proteins involved in splicing and leverage cysteine- and protein-directed activity-based protein profiling with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.

2024-10-21 | PXD056704 | Pride

RNA-Seq to Explore 2-Sulfonylpyrimidine Warheads as Acrylamide Surrogates for Targeted Covalent Inhibition of BTK in Diffuse Large B Cell (DLBCL) Cell Line TMD8

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Project description:A large swath of the human proteome is dedicated to RNA homeostasis, but most RNA-binding proteins lack chemical probes1,2. Here, phenotypic screening led to the discovery of electrophilic small molecules that swiftly (within 4 h) and stereospecifically decrease transcripts encoding the androgen receptor (AR) and its major V7 splice variant in human prostate cancer cells. We show by chemical proteomics that these compounds covalently engage cysteine-145 on the RNA-binding protein NONO. Transcriptomics and proteomics profiling revealed that covalent NONO ligands suppress a discrete set of transcripts and proteins, including multiple oncogenic transcription factors, and impair the proliferation of cancer cells. These effects were not observed following genetic disruption of NONO, which instead blocked ligand activity. The covalent ligands promote accumulation of NONO in nuclear foci and at the first 5 splice site of immature transcripts, pointing to a trapping mechanism that may prevent compensatory action by the related protein PSPC1, which was found to increase in cancer cells following genetic or chemical perturbation of NONO. These findings, taken together, designate NONO as a druggable RNA-binding protein that can be co-opted by covalent small molecules to suppress pro-tumorigenic transcriptional networks.

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Remodeling of oncogenic transcriptomes by small molecules targeting the RNA-binding protein NONO

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