ABSTRACT
Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery/methods , Molecular Targeted Therapy , Protein Kinase Inhibitors/pharmacology , Proteomics/methods , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cytokines/metabolism , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/enzymology , Lung Neoplasms/drug therapy , Lung Neoplasms/enzymology , Mice , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , Xenograft Model Antitumor Assays , fms-Like Tyrosine Kinase 3/antagonists & inhibitorsABSTRACT
Nucleotide-binding and oligomerization domain (NOD)-like receptors constitute a first line of defense against invading bacteria. X-linked Inhibitor of Apoptosis (XIAP) is implicated in the control of bacterial infections, and mutations in XIAP are causally linked to immunodeficiency in X-linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly complex (LUBAC) to NOD2. We further show that LUBAC activity is required for efficient NF-κB activation and secretion of proinflammatory cytokines after NOD2 stimulation. Remarkably, XLP-2-derived XIAP variants have impaired ubiquitin ligase activity, fail to ubiquitylate RIPK2, and cannot facilitate NOD2 signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis.
Subject(s)
Immunity, Innate , Inflammation/immunology , Nod2 Signaling Adaptor Protein/metabolism , X-Linked Inhibitor of Apoptosis Protein/genetics , Animals , Female , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nod2 Signaling Adaptor Protein/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Signal Transduction , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
Dendritic cell (DC)-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN: CD209) is a C-type lectin that binds ICAM-2,3 and various pathogens such as HIV, helicobacter, and mycobacteria. It has been suggested that Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, interacts with DC-SIGN to evade the immune system. To directly analyze the role of human DC-SIGN during mycobacterial infection, we generated conventional transgenic (tg) mice (termed "hSIGN") using CD209 cDNA under the control of the murine CD11c promoter. Upon mycobacterial infection, DCs from hSIGN mice produced significantly less IL-12p40 and no significant differences were be observed in the secretion levels of IL-10 relative to control DCs. After high dose aerosol infection with the strain M. tuberculosis H37Rv, hSIGN mice showed massive accumulation of DC-SIGN(+) cells in infected lungs, reduced tissue damage and prolonged survival. Based on our in vivo data, we propose that instead of favoring the immune evasion of mycobacteria, human DC-SIGN may have evolved as a pathogen receptor promoting protection by limiting tuberculosis-induced pathology.
