Protein Kinase CK2 Controls the Fate Between Th17 Cell and Regulatory T Cell Differentiation
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ABSTRACT: We report that CK2 inhibition with CX-4945 broadly suppresses Th17-associated effector and metabolic gene expression. CD4+ T cells were cultured in IL-6, IL-23 and TGFb1 for 72 h, in the absence of presence of 2 mM CX-4945, RNA-seq was performed and gene expression compared between the control and treatment groups.
Project description:High-risk B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive disease, often characterized by resistance to chemotherapy. A frequent feature of high-risk B-ALL is loss of function of the Ikaros tumor suppressor. In leukemia, Ikaros’ function is impaired by oncogenic Casein Kinase II (CK2), which is overexpressed in B-ALL. Phosphorylation by CK2 reduces Ikaros binding to the promoter of its target gene, particularly Bcl-xL. This results in a loss of Ikaros-mediated repression of Bcl-xL and in increased expression of Bcl-xL. Increased expression of Bcl-xL and/or CK2, as well as reduced Ikaros expression, are associated with resistance to doxorubicin treatment. Molecular and pharmacological inhibition of CK2 with a specific inhibitor CX-4945, enhances Ikaros-mediated repression of Bcl-xL and increases sensitivity to doxorubicin. Combination treatment with CX-4945 and doxorubicin show synergistic therapeutic effects in vitro and in preclinical models of high-risk B-ALL. Results reveal a novel signaling network that regulates chemoresistance in leukemia and lays the groundwork for clinical testing of CK2 inhibitors in combination with doxorubicin for the treatment of hematopoietic malignancies.
Project description:Specificity is a limiting factor when using small-molecule inhibitors to study protein kinase signalling. Since inhibitor-resistant kinase mutants (i.e., drug-resistant alleles) remain active in the presence of inhibitor, they facilitate validation of on-target effects. By combining an inhibitor-resistant kinase mutant with mass spectrometry-based phosphoproteomics, we previously devised a systematic strategy for reliable identification and validation of CSNK2 substrates. In this study, we use the same strategy to evaluate the selectivity of CX-4945, a clinical stage CSNK2 inhibitor, and SGC-CK2-1, a chemical probe selectively targeting CSNK2. Human osteosarcoma (U2OS) cells expressing exogenous wild-type CSNK2A1 (WT) or an inhibitor-resistant triple mutant (TM, V66A/H160D/I174A) were treated with CX-4945 or SGC-CK2-1 prior to analysis using triple SILAC (phospho)proteomics. The minority of phosphosites, 15% at 4 hours and 5% at 24 hours, that were significantly downregulated in response to CX-4945 treatment were determined to be CSNK2A1-dependent. By comparison, the majority of phosphosites, >55% at both 4 and 24 hours, that were significantly downregulated in response to SGC-CK2-1 were identified as CSNK2A1-dependent. This indicates that SGC-CK2-1 exhibits dramatically greater selectivity towards CSNK2A1 than CX-4945. Notably, utilization of SGC-CK2-1 in cells expressing TM-CSNK2A1 enabled the identification of 330 CSNK2A1-dependent phosphosites. Overall, this study highlights the utility of exploiting highly selective chemical probes together with inhibitor-resistant kinase mutants to identify bona fide kinase substrates.
Project description:Crohn’s disease (CD) is one of the major forms of inflammatory bowel disease (IBD), characterized by chronic inflammation of the gastrointestinal tract. CD is associated with aberrant Th1 and Th17 responses accompanied by high levels of IFN-g and IL-17, respectively. Protein kinase 2 (CK2) is a highly conserved serine-threonine kinase that is involved in several signal transduction pathways which regulate inflammatory responses. CK2 promotes Th17 cell differentiation and suppresses the generation of Foxp3+ regulatory T cells. The function of CK2 in CD4+ T-cells during the pathogenesis of CD is unknown. We utilized T-cell induced colitis model, transferring CD45RBhi naïve CD4+ T-cells from CK2afl/fl littermate control and CK2afl/fldLck-Cre mice into Rag1-/- mice. We demonstrate that CD4+ T-cells from CK2afl/fldLck-Cre mice fail to induce wasting disease and significant intestinal inflammation, which is associated with decreased IL-17A+, IFN-g+ and double positive IL-17A+ IFN-g+ CD4+ T-cells in the spleen and colon. Further, we determine that CK2a regulates CD4+ T-cell proliferation through a cell-intrinsic manner. CK2a is also important in controlling CD4+ T-cell responses by regulating NFAT2, which is vital for T-cell activation and proliferation. Thus, our data demonstrate that CK2a contributes to the pathogenesis of colitis by promoting CD4+ T-cell proliferation and Th1 and Th17 responses, and that targeting CK2 kinase activity may be a novel therapeutic treatment for CD patients.
Project description:Multicellular Tumor Spheroids (MCTS) were pre-formed for 3 days with 786-O cell line to better mimic the tumor microenviroenment. These spheroids were treated with either vehicle (DMSO), drugs alone (KU-60019 10 μM; CX-4945 5 μM) or in combination (KU + CX) for 48h before RNA extraction.
Project description:The early stages of human Th17 Cell differentiation were studied using label free proteomics to compare Th17 polarized CD4+ human T cells at 24 h and 72 h with activated cells (72 and 24 h) and Thp cells.
Project description:Interleukin 17 (IL-17) producing T helper 17 (Th17) cells are critical drivers of pathogenesis in a variety of autoimmune and inflammatory diseases. Strategies to mitigate excessive Th17 response thus remain an attractive target for immunotherapies. Here we report that Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) regulates IL-17 production by Th17 cells in human and mouse. Using CIP2A knock-out (KO) mice and siRNA-mediated CIP2A silencing in human primary CD4+ T cells, we demonstrated that CIP2A silencing results in a significant increase in IL-17 production. Interestingly, CIP2A deficient Th17 cells were characterized by increased strength and duration of STAT3 (Y705) phosphorylation. Genome-wide gene expression profile as well as the p-STAT3 (Y705) interactome of CIP2A deficient Th17 cells identified that CIP2A regulates the strength of the interaction between Acylglycerol kinase (AGK) and STAT3, and thereby, modulates STAT3 phosphorylation as well as expression of IL-17 in Th17 cells. Our results uncover the physiological function of CIP2A in Th17 cells and provides new opportunities for therapeutic intervention in Th17 cell mediated diseases.
Project description:Interleukin 23 (IL-23) triggers pathogenic features in pro-inflammatory, IL-17-secreting T cells (Th17 and Tγδ17) that play a key role in the development of inflammatory diseases. However, the IL-23 signaling cascade remains largely undefined. Here we used quantitative phosphoproteomics to characterize IL-23 signaling in primary murine Th17 cells. We quantified 6,888 phosphorylation sites in Th17 cells, and found 168 phosphorylations regulated upom IL-23 stimulation. IL-23 increased the phosphorylation of the myosin regulatory light chain (RLC), an actomyosin contractibility marker, in Th17 and Tγδ cells. IL-23-induced RLC phosphorylation required JAK2 and ROCK catalytic activity, and the study of the IL-23/ROCK axis revealed an unexpected role of IL-23 in the migration of Tγδ17 and Th17 cells. Moreover, pharmacological inhibition of ROCK reduced Tγδ17 recruitment to inflamed skin upon challenge with inflammatory agent Imiquimod. This work: i) provides new insights into phosphorylation networks that control Th17 cells, ii) widely expands the current knowledge on IL-23 signaling, and iii) contributes to the increasing list of immune cells subsets characterized by global phosphoproteomic approaches.
Project description:Th17 cells are highly proinflammatory cells that are critical for clearing extracellular pathogens like fungal infections and for induction of multiple autoimmune diseases1. IL-23 plays a critical role in stabilizing and endowing Th17 cells with pathogenic effector functions2. Previous studies have shown that IL-23 signaling reinforces the Th17 phenotype by increasing expression of IL-23 receptor (IL-23R)3. However, the precise molecular mechanism by which IL-23 sustains the Th17 response and induces pathogenic effector functions has not been elucidated. Here, we used unbiased transcriptional profiling of developing Th17 cells to construct a model of their signaling network and identify major nodes that regulate Th17 development. We identified serum glucocorticoid kinase-1 (SGK1), as an essential node downstream of IL-23 signaling, critical for regulating IL-23R expression and for stabilizing the Th17 cell phenotype by deactivation of Foxo1, a direct repressor of IL-23R expression. A serine-threonine kinase homologous to AKT4, SGK1 has been associated with cell cycle and apoptosis, and has been shown to govern Na+ transport and homeostasis5, 6 7, 8. We here show that a modest increase in salt (NaCl) concentration induces SGK1 expression, promotes IL-23R expression and enhances Th17 cell differentiation in vitro and in vivo, ultimately accelerating the development of autoimmunity. The loss of SGK1 resulted in abrogation of Na+-mediated Th17 differentiation in an IL-23-dependent manner. These data indicate that SGK1 is a critical regulator for the induction of pathogenic Th17 cells and provides a molecular insight by which an environmental factor such as a high salt diet could trigger Th17 development and promote tissue inflammation. Effects of NaCl on Th17 differentiation
Project description:Genome-wide DNA methylation profiling of human B-ALL cell line SEM after 48 h incubation with DMSO (control), CX-4945 (Silmitasertib), Decitabine (DEC) or combined CX-4945 + DEC treatment. The Infinium MethylationEPIC BeadChip was used to obtain DNA methylation profiles across approximately 866,895 CpG islands.