Project description:We investigated whether mouse serum autoantibody binding patterns on random-sequence peptide microarrays (immunosignaturing) can be used for diagnosing and predicting the onset of lupus and its central nervous system (CNS) manifestations. Submitter states "We have no processed data to submit. We have no gpr files to submit."
Project description:In addition to determining possible diagnostic and predictive peptides of lupus and CNS-lupus, we also used our microarray technology along with the Guitope computer program to determine possible natural protein match to five monoclonal autoantibodies that were created using one of the autoimmune MRL/lpr mouse. Submitter states "We have no processed data to submit. We have no gpr files to submit."
Project description:Cdk7, the CDK-activating kinase and transcription factor IIH component, is a target of inhibitors that kill cancer cells by exploiting tumor-specific transcriptional dependencies. However, whereas selective inhibition of analog-sensitive (AS) Cdk7 in colon cancer derived cells arrests division and disrupts transcription, it does not by itself trigger apoptosis efficiently. Here we show that p53 activation by 5-fluorouracil or nutlin-3 synergizes with a reversible Cdk7as inhibitor to induce cell death. Synthetic lethality was recapitulated with covalent inhibitors of wild-type Cdk7, THZ1 or the more selective YKL-1-116. The effects were allele-specific; a CDK7as mutation conferred both sensitivity to bulky adenine analogs and resistance to covalent inhibitors. Non-transformed colon epithelial cells were resistant to these combinations, as were cancer-derived cells with p53-inactivating mutations. Apoptosis was dependent on death receptor DR5, a p53 transcriptional target whose expression was refractory to Cdk7 inhibition. Therefore, p53 activation induces transcriptional dependency to sensitize cancer cells to Cdk7 inhibition.
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.
Project description:Bromodomain (BRD) is an epigenetic reader of acetylated lysine. It has emerged as a therapeutic target for cancer and other diseases. Current nonselective BRD inhibitors (BRDis) face several adverse events (i.e. gastrointestinal toxicity and thrombocytopenia), making the development of target covalent inhibitor (TCI) for BD1/2 as a fresh avenue to overcome safety challenges. We report herein a set of activity-based probes (ABPs; P3-P7) based on various lysine-reactive covalent warheads capable of global profiling of ligandable lysine within BRDs in live cells and animals. Chemoproteomic experiments with P7 by utilizing 2-ethynylbenzaldehyde (EBA) identified 16 endogenous BRDs, thus giving a global landscape of ligandable lysines in BRDs. By further introducing EBA and salicylaldehyde into PLX51107 (a non-covalent BRDi), we generated novel irreversible (BDS1-4) and reversible (BDS5-6) lysine-reactive TCIs. Mass spectrometry and X-ray crystallography confirmed the successful covalent engagement between the covalent warhead and lysine near Kac binding site. BDS4 showed 104-fold selectivity for BD1 over BD2 with prolonged anti-cancer effect than non-covalent BRDi. Importantly, BDS4 retained robust activity against fibrosis in cells and animals in comparison to the marginal effect of BD2 inhibitor RVX-208. Our work serves as a useful tool to delineate the distinct function of BD1 and BD2.
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, MM1S, Loucy, and HeLa (WT and Dox-inducible CDK7 mutant) 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.
Project description:The goal of this project was to use differential kinobeads profiling for the discrimination of reversible and irreversible kinase targets of different covalent kinase inhibitors. For this, differences in target engagement measured in a lysate- and a cell-based kinobeads assay were compared in a kinome-wide scale.