Covalent Targeting of Splicing in T Cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1171002 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR30936312.fastq.gz | Fastqsanger.gz | |||
| SRR30936313.fastq.gz | Fastqsanger.gz | |||
| SRR30936314.fastq.gz | Fastqsanger.gz | |||
| SRR30936315.fastq.gz | Fastqsanger.gz | |||
| SRR30936316.fastq.gz | Fastqsanger.gz |
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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 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.
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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.
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Project description:Cyclin-dependent kinase 7 (CDK7) plays a critical role in the general regulation of RNA polymerase II-mediated transcription. However, the absence of selective CDK7 inhibitors has hindered the ability to investigate the consequences of acute and prolonged inhibition of CDK7 under normal and pathological conditions. Here we present the discovery and characterization of the first covalent CDK7 inhibitor, CDK7-IN-1, that has the unprecedented ability to target a unique cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7 amongst the 20 known CDKs. Cancer cell line profiling indicates that a subset of cancer cell lines, including T-cell acute lymphoblastic leukemia (T-ALL), exhibit 100-fold greater sensitivity to CDK7-IN-1 over other tumor and normal cell lines. Genome-wide expression analysis in Jurkat T-ALL indicates that CDK7-IN-1 disproportionally affects RUNX1 as well as other components of the TAL1 transcriptional network and its targets, downregulating key regulators of transcription and apoptosis critical for the T-ALL state. These oncogenes are encoded by short-lived mRNA transcripts, are associated with super-enhancers, and exhibit a strong dependency on continuous transcription for sustained expression. Therefore, pharmacological modulation of CDK7 kinase activity may define a method for the identification and treatment of tumor types exhibiting extreme dependencies on transcription for maintenance of the oncogenic state. Jurkat cells were treated with various drugs including a covalent inhibitor of CDK7 (CDK7-IN-1), a reversible inhibitor of CDK7 (CDK7-IN-1), Flavopiridol, Actinomycin D, and DMSO controls. Replicates are annotated.
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