ABSTRACT
It has been shown that PRMT5 inhibition by small molecules can selectively kill cancer cells with homozygous deletion of the MTAP gene if the inhibitors can leverage the consequence of MTAP deletion, namely, accumulation of the MTAP substrate MTA. Herein, we describe the discovery of TNG908, a potent inhibitor that binds the PRMT5·MTA complex, leading to 15-fold-selective killing of MTAP-deleted (MTAP-null) cells compared to MTAPintact (MTAP WT) cells. TNG908 shows selective antitumor activity when dosed orally in mouse xenograft models, and its physicochemical properties are amenable for crossing the blood-brain barrier (BBB), supporting clinical study for the treatment of both CNS and non-CNS tumors with MTAP loss.
Subject(s)
Antineoplastic Agents , Protein-Arginine N-Methyltransferases , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/metabolism , Humans , Animals , Mice , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/chemical synthesis , Drug Discovery , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , Cell Line, Tumor , Xenograft Model Antitumor Assays , Neoplasms/drug therapy , Brain/metabolism , Structure-Activity RelationshipABSTRACT
14-3-3 proteins play critical roles in controlling multiple aspects of the cellular response to stress and DNA damage including regulation of metabolism, cell cycle progression, cell migration, and apoptotic cell death by binding to protein substrates of basophilic protein kinases following their phosphorylation on specific serine/threonine residues. Although over 200 mammalian proteins that bind to 14-3-3 have been identified, largely through proteomic studies, in many cases the relevant protein kinase responsible for conferring 14-3-3-binding to these proteins is not known. To facilitate the identification of kinase-specific 14-3-3 clients, we developed a biochemical approach using high-density protein filter arrays and identified the translational regulatory molecule PABPC1 as a substrate for Chk1 and MAPKAP Kinase-2 (MK2) in vitro, and for MK2 in vivo, whose phosphorylation results in 14-3-3-binding. We identify Ser-470 on PABPC1 within the linker region connecting the RRM domains to the PABC domain as the critical 14-3-3-binding site, and demonstrate that loss of PABPC1 binding to 14-3-3 results in increased cell proliferation and decreased cell death in response to UV-induced DNA damage.
ABSTRACT
Dysregulated PI3K/Akt/mTOR (PAM) pathway signaling occurs in ~30% of human cancers, making it a rational target for new therapies; however, the effectiveness of some PAM pathway inhibitors, such as mTORC rapalogs, may be compromised by a compensatory feedback loop leading to Akt activation. In this study, the p70S6K/Akt dual inhibitor, M2698 (previously MSC2363318A), was characterized as a potential anti-cancer agent through examination of its pharmacokinetic, pharmacodynamic and metabolic properties, and anti-tumor activity. M2698 was highly potent in vitro (IC50 1 nM for p70S6K, Akt1 and Akt3 inhibition; IC50 17 nM for pGSK3ß indirect inhibition) and in vivo (IC50 15 nM for pS6 indirect inhibition), and relatively selective (only 6/264 kinases had an IC50 within 10-fold of p70S6K). Orally administered M2698 crossed the blood-brain barrier in rats and mice, with brain tumor exposure 4-fold higher than non-disease brain. Dose-dependent inhibition of target substrate phosphorylation was observed in vitro and in vivo, indicating that M2698 blocked p70S6K to provide potent PAM pathway inhibition while simultaneously targeting Akt to overcome the compensatory feedback loop. M2698 demonstrated dose-dependent tumor growth inhibition in mouse xenograft models derived from PAM pathway-dysregulated human triple-negative (MDA-MB-468) and Her2-expressing breast cancer cell lines (MDA-MB-453 and JIMT-1), and reduced brain tumor burden and prolonged survival in mice with orthotopically implanted U251 glioblastoma. These findings highlight M2698 as a promising PAM pathway inhibitor whose unique mechanism of action and capacity to pass the blood-brain barrier warrant clinical investigation in cancers with PAM pathway dysregulation, and those with central nervous system involvement.
ABSTRACT
The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK in vivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21-activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism's response to low nutrients during development, or in adult stem and cancer cells.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Gene Expression Regulation, Enzymologic/genetics , Mitosis/genetics , AMP-Activated Protein Kinases/genetics , Adenosine Triphosphate/metabolism , Cell Line, Tumor , HEK293 Cells , Humans , Myosin Light Chains/metabolism , Phosphorylation , Protein Phosphatase 1/genetics , Protein Phosphatase 1/metabolism , Substrate Specificity , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolismABSTRACT
Isoelectric focusing (IEF) is the first step for two-dimensional (2D) gel electrophoresis and plays an important role in sample purification for proteomics. However, biases in protein size and pI resolution, as well as limitations in sample volume, gel capacity, sample loss, and experimental time, remain challenges. In order to address some of the limitations of traditional IEF, we present a microfluidic free flow IEF (FF-IEF) device for continuous protein separation into 24 fractions. The device reproducibly establishes a nearly linear pH gradient from 4 to 10. Optimized dynamic coatings of 4% poly(vinyl alcohol) (PVA) minimize peak broadening by transverse electrokinetic flows. Even though the device operates at high electric fields (up to 370 V/cm), efficient cooling maintains solution temperature inside the separation channel controllably in the range 2-25 degrees C. Protein samples with a dynamic concentration range from microg/mL to mg/mL can be loaded into the microdevice at a flow rate of 1 mL/h and residence time of approximately 12 min. By using a protein complex of nine proteins and 13 isoforms, we demonstrate improved separation with the FF-IEF system over traditional 2D gel electrophoresis. Device-to-device reproducibility is also illustrated through the efficient depletion of the albumin and hemoglobin assays. Post-device sample concentrations result in a 10-20-fold increase, which allow for isolation and detection of low abundance proteins. The separation of specific proteins from a whole cell lysate is demonstrated as an example. The microdevice has the further benefits of retaining high molecular weight proteins, providing higher yield of protein that has a broader range in pI, and reducing experimental time compared to conventional IEF IGP gel strip approaches.
Subject(s)
Isoelectric Focusing/instrumentation , Microfluidic Analytical Techniques , Proteins/isolation & purification , Albumins/analysis , Albumins/isolation & purification , Cell Extracts/chemistry , Electroosmosis , HeLa Cells , Hemoglobins/analysis , Hemoglobins/isolation & purification , Humans , Hydrogen-Ion Concentration , Isoelectric Focusing/methods , Proteins/analysis , Reproducibility of Results , TemperatureABSTRACT
Neurofibromatosis type 2 (NF2) is a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate Rac1 signaling by inhibiting its downstream effector kinases, the p21-activated kinases (Pak). Given the implication of Paks in tumorigenesis, it is plausible that merlin's tumor suppressive function might be mediated, at least in part, via inhibition of the Paks. We present data indicating this is indeed the case. First, analysis of primary schwannoma samples derived from NF2 patients showed that in a significant fraction of the tumors, the activity of Pak1 was highly elevated. Second, we used shRNAs to knockdown Pak1, 2, and 3 in NIH3T3 cells expressing a dominant-negative form of merlin, NF2(BBA) (NIH3T3/NF2(BBA)), and find that simultaneous knockdown of Pak1-3 in these cells significantly reduced their growth rates in vitro and inhibited their ability to form tumors in vivo. Finally, while attempting to silence Pak1 in rat schwannoma cells, we found that these cells were unable to tolerate long-term Pak1 inhibition and rapidly moved to restore Pak1 levels by shutting down Pak1 shRNA expression through a methylation-dependent mechanism. These data suggest that inhibiting Pak could be a beneficial approach for the development of therapeutics toward NF2. In addition, the finding that the shRNA-mediated Pak1 suppression was silenced rapidly by methylation raises questions about the future application of such technologies for the treatment of diseases such as cancer.
Subject(s)
Neurilemmoma/enzymology , Neurofibromatosis 2/enzymology , p21-Activated Kinases/antagonists & inhibitors , Animals , Base Sequence , Brain Neoplasms/enzymology , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Proliferation/drug effects , Cells, Cultured , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Mice , Molecular Sequence Data , NIH 3T3 Cells , Neurilemmoma/genetics , Neurilemmoma/pathology , Neurofibromatosis 2/genetics , Neurofibromatosis 2/pathology , RNA, Small Interfering/pharmacology , Transplantation, Heterologous , p21-Activated Kinases/genetics , p21-Activated Kinases/physiologyABSTRACT
14-3-3 proteins are crucial in a wide variety of cellular responses including cell cycle progression, DNA damage checkpoints and apoptosis. One particular 14-3-3 isoform, sigma, is a p53-responsive gene, the function of which is frequently lost in human tumours, including breast and prostate cancers as a result of either hypermethylation of the 14-3-3sigma promoter or induction of an oestrogen-responsive ubiquitin ligase that specifically targets 14-3-3sigma for proteasomal degradation. Loss of 14-3-3sigma protein occurs not only within the tumours themselves but also in the surrounding pre-dysplastic tissue (so-called field cancerization), indicating that 14-3-3sigma might have an important tumour suppressor function that becomes lost early in the process of tumour evolution. The molecular basis for the tumour suppressor function of 14-3-3sigma is unknown. Here we report a previously unknown function for 14-3-3sigma as a regulator of mitotic translation through its direct mitosis-specific binding to a variety of translation/initiation factors, including eukaryotic initiation factor 4B in a stoichiometric manner. Cells lacking 14-3-3sigma, in marked contrast to normal cells, cannot suppress cap-dependent translation and do not stimulate cap-independent translation during and immediately after mitosis. This defective switch in the mechanism of translation results in reduced mitotic-specific expression of the endogenous internal ribosomal entry site (IRES)-dependent form of the cyclin-dependent kinase Cdk11 (p58 PITSLRE), leading to impaired cytokinesis, loss of Polo-like kinase-1 at the midbody, and the accumulation of binucleate cells. The aberrant mitotic phenotype of 14-3-3sigma-depleted cells can be rescued by forced expression of p58 PITSLRE or by extinguishing cap-dependent translation and increasing cap-independent translation during mitosis by using rapamycin. Our findings show how aberrant mitotic translation in the absence of 14-3-3sigma impairs mitotic exit to generate binucleate cells and provides a potential explanation of how 14-3-3sigma-deficient cells may progress on the path to aneuploidy and tumorigenesis.
Subject(s)
Biomarkers, Tumor/metabolism , Cytokinesis , Exonucleases/metabolism , Mitosis , Neoplasm Proteins/metabolism , Protein Biosynthesis , 14-3-3 Proteins , Biomarkers, Tumor/deficiency , Cell Line, Tumor , Eukaryotic Initiation Factors/metabolism , Exonucleases/deficiency , Exoribonucleases , Gene Expression Regulation, Neoplastic , Humans , Neoplasm Proteins/deficiency , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , RNA Caps/genetics , RNA Caps/metabolismABSTRACT
Glioblastoma multiforme is the most common and lethal form of primary brain cancer. Diagnosis of this advanced glioma has a poor prognosis due to the ineffectiveness of current therapies. Aberrant expression of receptor tyrosine kinases (RTK) in glioblastoma multiformes is suggestive of their role in initiation and maintenance of these tumors of the central nervous system. In fact, ectopic expression of the orphan RTK ROS is a frequent event in human brain cancers, yet the pathologic significance of this expression remains undetermined. Here, we show that a glioblastoma-associated, ligand-independent rearrangement product of ROS (FIG-ROS) cooperates with loss of the tumor suppressor gene locus Ink4a;Arf to produce glioblastomas in the mouse. We show that this FIG-ROS-mediated tumor formation in vivo parallels the activation of the tyrosine phosphatase SH2 domain-containing phosphatase-2 (SHP-2) and a phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling axis in tumors and tumor-derived cell lines. We have established a fully penetrant preclinical model for adult onset of glioblastoma multiforme in keeping with major genetic events observed in the human disease. These findings provide novel and important insights into the role of ROS and SHP-2 function in solid tumor biology and set the stage for preclinical testing of targeted therapeutic approaches.
Subject(s)
Brain Neoplasms/metabolism , Glioblastoma/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Astrocytoma/enzymology , Astrocytoma/metabolism , Astrocytoma/pathology , Brain Neoplasms/enzymology , Brain Neoplasms/pathology , Cyclin-Dependent Kinase Inhibitor p16/deficiency , Cyclin-Dependent Kinase Inhibitor p16/genetics , Enzyme Activation , Glioblastoma/enzymology , Glioblastoma/pathology , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Protein Phosphatase 2 , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatases/metabolism , Protein-Tyrosine Kinases/biosynthesis , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SH2 Domain-Containing Protein Tyrosine Phosphatases , Signal Transduction , TOR Serine-Threonine Kinases , Tumor Suppressor Protein p14ARF/deficiency , Tumor Suppressor Protein p14ARF/genetics , src Homology DomainsABSTRACT
The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3sigma, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3sigma-specific functions is unknown. We demonstrate that endogenous 14-3-3sigma preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3sigma bound to an optimal phosphopeptide ligand at 2.4 angstroms resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3sigma homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3sigma-containing heterodimers, rationalizing preferential homodimerization of 14-3-3sigma in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3sigma. Instead, the structure suggests a second ligand binding site involved in 14-3-3sigma-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three sigma-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3sigma the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to sigma.
Subject(s)
Biomarkers, Tumor/physiology , Exonucleases/physiology , Neoplasm Proteins/physiology , 14-3-3 Proteins , Amino Acid Sequence , Animals , Biomarkers, Tumor/metabolism , Blotting, Western , Cell Cycle Proteins/chemistry , Cell Line, Tumor , Crystallography, X-Ray , DNA Damage , Dimerization , Electrophoresis, Gel, Two-Dimensional , Exonucleases/metabolism , Exoribonucleases , Genetic Vectors , Humans , Immunoprecipitation , Ligands , Models, Molecular , Molecular Sequence Data , Mutation , Neoplasm Proteins/metabolism , Phylogeny , Protein Binding , Protein Conformation , Protein Isoforms , Sequence Homology, Amino Acid , Signal Transduction , Substrate Specificity , Transfection , cdc25 Phosphatases/chemistryABSTRACT
The Nf2 tumor suppressor gene codes for merlin, a protein whose function has been elusive. We describe a novel interaction between merlin and p21-activated kinase 1 (Pak1), which is dynamic and facilitated upon increased cellular confluence. Merlin inhibits the activation of Pak1, as the loss of merlin expression results in the inappropriate activation of Pak1 under conditions associated with low basal activity. Conversely, the overexpression of merlin in cells that display a high basal activity of Pak1 resulted in the inhibition of Pak1 activation. This inhibitory function of merlin is mediated through its binding to the Pak1 PBD and by inhibiting Pak1 recruitment to focal adhesions. This link provides a possible mechanism for the effect of loss of merlin expression in tumorigenesis.