RESUMO
During microbial infections, plasmacytoid dendritic cells (pDCs) are a main source of type I interferons α/ß (IFN-α/-ß). Nucleic acids from microbes are sensed by Toll-like receptors 7/9 (TLR7/9), which are selectively expressed in pDCs. Activated pDCs also produce proinflammatory cytokines and upregulate costimulatory molecules. Together, this equips pDCs with the ability to prime T, B, and NK cells and conventional DCs, thereby initiating adaptive immune responses. To avoid deleterious effects to the host, tight regulation of pDC activation is required. Despite data linking aberrant activation of pDCs with autoimmune diseases, little is known about mechanisms controlling pDC activation. Here, we investigated the role of microRNA-146a (miR-146a) in TLR pathway regulation in human pDCs. MiR-146a expression was induced upon TLR7/9 signaling. Furthermore, ectopic miR-146a expression effectively impaired TLR-mediated signaling in pDCs as TLR-induced nuclear factor-κB activation was reduced. This consequently diminished the production of proinflammatory cytokines and reduced pDC survival. Moreover, miR-146a-expressing pDCs had decreased ability to induce CD4(+) T-cell proliferation likely due to reduced expression levels of major histocompatibility complex class II and costimulatory molecules. Our data unravel the crucial immunomodulatory role of miR-146a in pDCs and may add to our understanding of aberrant responses in autoimmune diseases.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , MicroRNAs/farmacologia , Imunidade Adaptativa , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Pré-Escolar , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Lactente , Ativação Linfocitária , NF-kappa B/genética , NF-kappa B/imunologia , Transdução de Sinais , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologiaRESUMO
Plasmacytoid dendritic cells (pDCs) selectively express Toll-like receptor (TLR)-7 and TLR-9, which allow them to rapidly secrete massive amounts of type I interferons after sensing nucleic acids derived from viruses or bacteria. It is not completely understood how development and function of pDCs are controlled at the transcriptional level. One of the main factors driving pDC development is the ETS factor Spi-B, but little is known about its target genes. Here we demonstrate that Spi-B is crucial for the differentiation of hematopoietic progenitor cells into pDCs by controlling survival of pDCs and its progenitors. In search for Spi-B target genes, we identified the antiapoptotic gene Bcl2-A1 as a specific and direct target gene, thereby consolidating the critical role of Spi-B in cell survival.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células Dendríticas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Plasmócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Transcrição/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Pré-Escolar , Proteínas de Ligação a DNA/genética , Células Dendríticas/citologia , Feminino , Células-Tronco Hematopoéticas/citologia , Humanos , Lactente , Masculino , Antígenos de Histocompatibilidade Menor , Plasmócitos/citologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fatores de Transcrição/genéticaRESUMO
The spindle assembly checkpoint kinase TTK (Mps1) is a key regulator of chromosome segregation and is the subject of novel targeted therapy approaches by small-molecule inhibitors. Although the first TTK inhibitors have entered phase I dose escalating studies in combination with taxane chemotherapy, a patient stratification strategy is still missing. With the aim to identify a genomic biomarker to predict the response of tumor cells to TTK inhibitor therapy, we profiled a set of preclinical and clinical TTK inhibitors from different chemical series on a panel of 66 genetically characterized cell lines derived from different tumors (Oncolines). Cell lines harboring activating mutations in the CTNNB1 gene, encoding the Wnt pathway signaling regulator ß-catenin, were on average up to five times more sensitive to TTK inhibitors than cell lines wild-type for CTNNB1 The association of CTNNB1-mutant status and increased cancer cell line sensitivity to TTK inhibition was confirmed with isogenic cell line pairs harboring either mutant or wild-type CTNNB1 Treatment of a xenograft model of a CTNNB1-mutant cell line with the TTK inhibitor NTRC 0066-0 resulted in complete inhibition of tumor growth. Mutations in CTNNB1 occur at relatively high frequency in endometrial cancer and hepatocellular carcinoma, which are known to express high TTK levels. We propose mutant CTNNB1 as a prognostic drug response biomarker, enabling the selection of patients most likely to respond to TTK inhibitor therapy in proof-of-concept clinical trials. Mol Cancer Ther; 16(11); 2609-17. ©2017 AACR.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Proteínas de Ciclo Celular/genética , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , beta Catenina/genética , Animais , Biomarcadores Farmacológicos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Via de Sinalização Wnt/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The protein kinase threonine tyrosine kinase (TTK; also known as Mps1) is a critical component of the spindle assembly checkpoint and a promising drug target for the treatment of aggressive cancers, such as triple negative breast cancer. While the first TTK inhibitors have entered clinical trials, little is known about how the inhibition of TTK with small-molecule compounds affects cellular activity. We studied the selective TTK inhibitor NTRC 0066-0, which was developed in our own laboratory, together with 11 TTK inhibitors developed by other companies, including Mps-BAY2b, BAY 1161909, BAY 1217389 (Bayer), TC-Mps1-12 (Shionogi), and MPI-0479605 (Myrexis). Parallel testing shows that the cellular activity of these TTK inhibitors correlates with their binding affinity to TTK and, more strongly, with target residence time. TTK inhibitors are therefore an example where target residence time determines activity in in vitro cellular assays. X-ray structures and thermal stability experiments reveal that the most potent compounds induce a shift of the glycine-rich loop as a result of binding to the catalytic lysine at position 553. This "lysine trap" disrupts the catalytic machinery. Based on these insights, we developed TTK inhibitors, based on a (5,6-dihydro)pyrimido[4,5-e]indolizine scaffold, with longer target residence times, which further exploit an allosteric pocket surrounding Lys553. Their binding mode is new for kinase inhibitors and can be classified as hybrid Type I/Type III. These inhibitors have very potent anti-proliferative activity that rivals classic cytotoxic therapy. Our findings will open up new avenues for more applications for TTK inhibitors in cancer treatment.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas de Ciclo Celular/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/químicaRESUMO
Inhibition of the spindle assembly checkpoint kinase TTK causes chromosome mis-segregation and tumor cell death. However, high levels of TTK correlate with chromosomal instability (CIN), which can lead to aneuploidy. We show that treatment of tumor cells with the selective small molecule TTK inhibitor NTRC 0066-0 overrides the mitotic checkpoint, irrespective of cell line sensitivity. In stable aneuploid cells NTRC 0066-0 induced acute CIN, whereas in cells with high levels of pre-existing CIN there was only a small additional fraction of cells mis-segregating their chromosomes. In proliferation assays stable aneuploid cells were more sensitive than cell lines with pre-existing CIN. Tetraploids are thought to be an intermediate between diploid and unstable aneuploid cells. TTK inhibitors had the same potency on post-tetraploid and parental diploid cells, which is remarkable because the post-tetraploids are more resistant to mitotic drugs. Finally, we confirm that the reference compound reversine is a TTK inhibitor and like NTRC 0066-0, inhibits the proliferation of patient-derived colorectal cancer organoids. In contrast, treatment with TTK inhibitor did not reduce the viability of non-proliferating T cell acute lymphoblastic leukemia cells samples. Consequently, TTK inhibitor therapy is expected to spare non-dividing cells, and may be used to target stable aneuploid tumors.