ABSTRACT
The interaction of menin (MEN1) and MLL (MLL1, KMT2A) is a dependency and provides a potential opportunity for treatment of NPM1-mutant (NPM1mut) and MLL-rearranged (MLL-r) leukemias. Concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 occur in both leukemias and are particularly common in the NPM1mut subtype. In this study, transcriptional profiling after pharmacological inhibition of the menin-MLL complex revealed specific changes in gene expression, with downregulation of the MEIS1 transcription factor and its transcriptional target gene FLT3 being the most pronounced. Combining menin-MLL inhibition with specific small-molecule kinase inhibitors of FLT3 phosphorylation resulted in a significantly superior reduction of phosphorylated FLT3 and transcriptional suppression of genes downstream of FLT3 signaling. The drug combination induced synergistic inhibition of proliferation, as well as enhanced apoptosis, compared with single-drug treatment in models of human and murine NPM1mut and MLL-r leukemias harboring an FLT3 mutation. Primary acute myeloid leukemia (AML) cells harvested from patients with NPM1mutFLT3mut AML showed significantly better responses to combined menin and FLT3 inhibition than to single-drug or vehicle control treatment, whereas AML cells with wild-type NPM1, MLL, and FLT3 were not affected by either of the 2 drugs. In vivo treatment of leukemic animals with MLL-r FLT3mut leukemia reduced leukemia burden significantly and prolonged survival compared with results in the single-drug and vehicle control groups. Our data suggest that combined menin-MLL and FLT3 inhibition represents a novel and promising therapeutic strategy for patients with NPM1mut or MLL-r leukemia and concurrent FLT3 mutation.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Gene Expression Regulation, Leukemic/drug effects , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Leukemia, Myeloid, Acute/drug therapy , Myeloid-Lymphoid Leukemia Protein/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Coculture Techniques , Drug Synergism , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Mice, Inbred NOD , Myeloid Ecotropic Viral Integration Site 1 Protein/biosynthesis , Myeloid Ecotropic Viral Integration Site 1 Protein/genetics , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Nuclear Proteins/genetics , Nucleophosmin , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational , Random Allocation , Transcription, Genetic/drug effects , fms-Like Tyrosine Kinase 3/biosynthesis , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
The adult intestinal stem cells (ISCs) are transcriptionally heterogeneous. As the mechanisms governing their developmental specification are still poorly understood, whether this heterogeneity reflects an early determination of distinct cellular sub-types with potentially distinct physiological functions remains an open question. We investigate the cellular heterogeneity within the mouse embryonic midgut epithelium at the molecular and functional levels. Cell fate mapping analysis revealed that multiple early embryonic epithelial progenitors give rise to Lgr5+ ISCs. The origin of the molecularly distinct early precursors along the anterior-posterior axis defines the transcriptional signature of embryonic Lgr5+ ISC progenitors. We further show that the early epithelial progenitors have different capacity to generate Lgr5+ ISC progenitors and Axin2+ early precursors display the highest potential.
Subject(s)
Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Adult Stem Cells/physiology , Animals , Cell Differentiation , Digestive System , Embryonic Stem Cells/physiology , Endoderm , Epithelial Cells/metabolism , Intestinal Mucosa/metabolism , Intestines , Mice , Mice, Transgenic , Stem Cells/physiologyABSTRACT
The adult intestinal stem cells (ISCs), their hierarchies, mechanisms of maintenance and differentiation have been extensively studied. However, when and how ISCs are established during embryogenesis remains unknown. We show here that the transcription regulator Id2 controls the specification of embryonic Lgr5+ progenitors in the developing murine small intestine. Cell fate mapping analysis revealed that Lgr5+ progenitors emerge at E13.5 in wild-type embryos and differ from the rest on the intestinal epithelium by a characteristic ISC signature. In the absence of Id2, the intestinal epithelium differentiates into Lgr5+ cells already at E9.5. Furthermore, the size of the Lgr5+ cell pool is significantly increased. We show that Id2 restricts the activity of the Wnt signalling pathway at early stages and prevents precocious differentiation of the embryonic intestinal epithelium. Id2-deficient embryonic epithelial cells cultured ex vivo strongly activate Wnt target genes as well as markers of neoplastic transformation and form fast growing undifferentiated spheroids. Furthermore, adult ISCs from Id2-deficient mice display a distinct transcriptional signature, supporting an essential role for Id2 in the correct specification of ISCs.
Subject(s)
Inhibitor of Differentiation Protein 2/metabolism , Intestine, Small/embryology , Receptors, G-Protein-Coupled/analysis , Stem Cells/chemistry , Stem Cells/physiology , Animals , Mice , Wnt Signaling PathwayABSTRACT
YciH is a bacterial protein, homologous to eukaryotic translation initiation factor eIF1. Preceding evidence obtained with the aid of in vitro translation initiation system suggested that it may play a role of a translation initiation factor, ensuring selection against suboptimal initiation complexes. Here we studied the effect of Escherichia coli yciH gene inactivation on translation of model mRNAs. Neither the translation efficiency of leaderless mRNAs, nor mRNAs with non AUG start codons, was found to be affected by YciH in vivo. Comparative proteome analysis revealed that yciH gene knockout leads to a more than fold2- increase in expression of 66 genes and a more than fold2- decrease in the expression of 20 genes. Analysis of these gene sets allowed us to suggest a role of YciH as an inhibitor of translation in a stress response rather than the role of a translation initiation factor.
Subject(s)
Escherichia coli Proteins/metabolism , Eukaryotic Initiation Factors/metabolism , Gene Expression Regulation , Peptide Chain Initiation, Translational , Peptide Initiation Factors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Stress, Physiological/genetics , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli/metabolism , Protein Biosynthesis , ProteomeABSTRACT
Chemical landscape of natural RNA species is decorated with the large number of modified nucleosides. Some of those could easily be detected by reverse transcription, while others permit only high-performance liquid chromatography or mass-spectrometry detection. Presence of m(6)A nucleoside at a particular position of long RNA molecule is challenging to observe. Here we report an easy and high-throughput method for detection of m(6)A nucleosides in RNA based on high-resolution melting analysis. The method relies on the previous knowledge of the modified nucleoside position at a particular place of RNA and allows rapid screening for conditions or genes necessary for formation of that modification.
Subject(s)
Adenosine/chemistry , Nucleic Acid Hybridization/methods , RNA/chemistry , Adenosine/analysis , Adenosine/metabolism , HEK293 Cells , Humans , Methylation , Methyltransferases/genetics , Oligonucleotide Probes , RNA/metabolismABSTRACT
The ribosomal RNA (rRNA) of Escherichia coli contains 24 methylated residues. A set of 22 methyltransferases responsible for modification of 23 residues has been described previously. Herein we report the identification of the yhiR gene as encoding the enzyme that modifies the 23S rRNA nucleotide A2030, the last methylated rRNA nucleotide whose modification enzyme was not known. YhiR prefers protein-free 23S rRNA to ribonucleoprotein particles containing only part of the 50S subunit proteins and does not methylate the assembled 50S subunit. We suggest renaming the yhiR gene to rlmJ according to the rRNA methyltransferase nomenclature. The phenotype of yhiR knockout gene is very mild under various growth conditions and at the stationary phase, except for a small growth advantage at anaerobic conditions. Only minor changes in the total E. coli proteome could be observed in a cell devoid of the 23S rRNA nucleotide A2030 methylation.
