ABSTRACT
The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.
Subject(s)
Azepines/pharmacology , Drug Discovery , Leukemia, Megakaryoblastic, Acute/drug therapy , Megakaryocytes/metabolism , Polyploidy , Pyrimidines/pharmacology , Small Molecule Libraries , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Animals , Aurora Kinase A , Aurora Kinases , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Humans , Leukemia, Megakaryoblastic, Acute/genetics , Megakaryocytes/cytology , Megakaryocytes/pathology , Mice , Mice, Inbred C57BL , Protein Interaction Maps , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , rho-Associated Kinases/metabolismABSTRACT
Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis.
Subject(s)
Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 4/genetics , Bone Morphogenetic Protein 7/genetics , Limb Buds/embryology , Polydactyly/genetics , Animals , Apoptosis , Bone Morphogenetic Protein 2/antagonists & inhibitors , Bone Morphogenetic Protein 4/antagonists & inhibitors , Bone Morphogenetic Protein 7/antagonists & inhibitors , Bone Morphogenetic Proteins/antagonists & inhibitors , Bone Morphogenetic Proteins/genetics , Cell Differentiation/genetics , Cell Proliferation , Chondrogenesis/genetics , Cytokines , Fibroblast Growth Factors/metabolism , Gene Expression Regulation, Developmental , Hindlimb/embryology , Intercellular Signaling Peptides and Proteins/biosynthesis , Intercellular Signaling Peptides and Proteins/genetics , Mesoderm/embryology , Mice , Mice, Transgenic , Mutation , Polydactyly/embryology , SOX9 Transcription Factor/biosynthesis , Signal Transduction/geneticsABSTRACT
We report the discovery of small molecules that target the Rho pathway, which is a central regulator of cytokinesis--the final step in cell division. We have developed a way of targeting a small molecule screen toward a specific pathway, which should be widely applicable to the investigation of any signaling pathway. In a chemical genetic variant of a classical modifier screen, we used RNA interference (RNAi) to sensitize cells and identified small molecules that suppressed or enhanced the RNAi phenotype. We discovered promising candidate molecules, which we named Rhodblock, and we identified the target of Rhodblock as Rho kinase. Several Rhodblocks inhibited one function of the Rho pathway in cells: the correct localization of phosphorylated myosin light chain during cytokinesis. Rhodblocks differentially perturb Rho pathway proteins in cells and can be used to dissect the mechanism of the Rho pathway during cytokinesis.
Subject(s)
Cytokinesis/physiology , rho-Associated Kinases/metabolism , Animals , Cytokinesis/drug effects , Drosophila/enzymology , Drosophila/genetics , Drosophila/physiology , Drosophila Proteins/metabolism , Enzyme Inhibitors/pharmacology , GTP Phosphohydrolases/metabolism , GTPase-Activating Proteins/metabolism , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Humans , Image Enhancement , Kinetics , Myosin Type II/metabolism , Proto-Oncogene Proteins/metabolism , RNA/antagonists & inhibitors , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Signal Transduction , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/drug effectsABSTRACT
Visual analysis is required to perform many biological experiments, from counting colonies to measuring the size or fluorescence intensity of individual cells or organisms. This unit outlines the use of CellProfiler, a free, open-source image analysis tool that extracts quantitative information from biological images. It includes a step-by-step protocol for automated analysis of the number, color, and size of yeast colonies growing on agar plates, but the methods can be adapted to identify and measure many other types of objects in images. The flexibility of the software allows experimenters to create pipelines of adjustable modules to fit different biological experiments and to generate accurate measurements from dozens or even hundreds of thousands of images.
Subject(s)
Automation, Laboratory/methods , Image Processing, Computer-Assisted/methods , Culture Media/chemistry , Saccharomyces cerevisiae/growth & developmentABSTRACT
Visual analysis is required to perform many biological experiments, from counting yeast colonies to measuring the size and shape of individual cells or the intensity of fluorescently labeled proteins within them. This unit outlines the use of CellProfiler, a free, open-source image analysis tool that extracts quantitative information from biological images. It includes a step-by-step protocol for automated analysis of the number, color, and size of yeast colonies growing on agar plates, but the methods can be adapted to identify and measure any objects in images. The flexibility of the software allows users to tailor pipelines of adjustable modules to fit different biological experiments, to generate accurate measurements from dozens or even hundreds of thousands of images.