ABSTRACT
Pyruvate kinase (PK) deficiency is a rare genetic disease that causes chronic hemolytic anemia. There are currently no targeted therapies for PK deficiency. Here, we describe the identification and characterization of AG-348, an allosteric activator of PK that is currently in clinical trials for the treatment of PK deficiency. We demonstrate that AG-348 can increase the activity of wild-type and mutant PK enzymes in biochemical assays and in patient red blood cells treated ex vivo. These data illustrate the potential for AG-348 to restore the glycolytic pathway activity in patients with PK deficiency and ultimately lead to clinical benefit.
Subject(s)
Enzyme Activators/pharmacology , Enzyme Activators/therapeutic use , Erythrocytes/enzymology , Pyruvate Kinase/deficiency , Pyruvate Kinase/metabolism , Quinolines/pharmacology , Quinolines/therapeutic use , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , Anemia, Hemolytic, Congenital Nonspherocytic , Animals , Enzyme Activation/drug effects , Enzyme Activators/chemistry , Erythrocytes/drug effects , Humans , Kinetics , Mice , Piperazines , Pyruvate Kinase/drug effects , Pyruvate Metabolism, Inborn Errors , Quinolines/chemistry , Recombinant Proteins/metabolism , Sulfonamides/chemistry , Tissue DonorsABSTRACT
The most common congenital disorder of glycosylation (CDG), phosphomannomutase 2 (PMM2)-CDG, is caused by mutations in PMM2 that limit availability of mannose precursors required for protein N-glycosylation. The disorder has no therapy and there are no models to test new treatments. We generated compound heterozygous mice with the R137H and F115L mutations in Pmm2 that correspond to the most prevalent alleles found in patients with PMM2-CDG. Many Pmm2R137H/F115L mice died prenatally, while survivors had significantly stunted growth. These animals and cells derived from them showed protein glycosylation deficiencies similar to those found in patients with PMM2-CDG. Growth-related glycoproteins insulin-like growth factor (IGF) 1, IGF binding protein-3 and acid-labile subunit, along with antithrombin III, were all deficient in Pmm2R137H/F115L mice, but their levels in heterozygous mice were comparable to wild-type (WT) littermates. These imbalances, resulting from defective glycosylation, are likely the cause of the stunted growth seen both in our model and in PMM2-CDG patients. Both Pmm2R137H/F115L mouse and PMM2-CDG patient-derived fibroblasts displayed reductions in PMM activity, guanosine diphosphate mannose, lipid-linked oligosaccharide precursor and total cellular protein glycosylation, along with hypoglycosylation of a new endogenous biomarker, glycoprotein 130 (gp130). Over-expression of WT-PMM2 in patient-derived fibroblasts rescued all these defects, showing that restoration of mutant PMM2 activity is a viable therapeutic strategy. This functional mouse model of PMM2-CDG, in vitro assays and identification of the novel gp130 biomarker all shed light on the human disease, and moreover, provide the essential tools to test potential therapeutics for this untreatable disease.
Subject(s)
Biomarkers , Congenital Disorders of Glycosylation/genetics , Cytokine Receptor gp130/genetics , Phosphotransferases (Phosphomutases)/genetics , Animals , Congenital Disorders of Glycosylation/metabolism , Congenital Disorders of Glycosylation/pathology , Cytokine Receptor gp130/biosynthesis , Disease Models, Animal , Fibroblasts/metabolism , Gene Expression Regulation , Genotype , Glycosylation , Humans , Mannose/genetics , Mannose/metabolism , Mice , MutationABSTRACT
Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 allele-alcohol interaction may be an even greater human public health hazard than previously appreciated.
Subject(s)
Aldehyde Dehydrogenase/genetics , Carcinoma, Hepatocellular/enzymology , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , Mutation/genetics , Alcoholic Intoxication/enzymology , Alcoholic Intoxication/pathology , Aldehyde Dehydrogenase, Mitochondrial , Amino Acid Substitution , Animals , Base Sequence , Carcinogenesis/pathology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Ethanol/adverse effects , Gene Knock-In Techniques , Genotyping Techniques , Hepatocytes/enzymology , Hepatocytes/pathology , Humans , Hyperpigmentation/pathology , Immunohistochemistry , Liver/enzymology , Liver/pathology , Liver Neoplasms/pathology , Mice, Inbred C57BL , Mutant Proteins/metabolism , Polymorphism, Genetic , Proteasome Endopeptidase Complex/metabolism , Protein Stability , Skin/pathology , Survival AnalysisABSTRACT
Highly rearranged and mutated cancer genomes present major challenges in the identification of pathogenetic events driving the neoplastic transformation process. Here we engineered lymphoma-prone mice with chromosomal instability to assess the usefulness of mouse models in cancer gene discovery and the extent of cross-species overlap in cancer-associated copy number aberrations. Along with targeted re-sequencing, our comparative oncogenomic studies identified FBXW7 and PTEN to be commonly deleted both in murine lymphomas and in human T-cell acute lymphoblastic leukaemia/lymphoma (T-ALL). The murine cancers acquire widespread recurrent amplifications and deletions targeting loci syntenic to those not only in human T-ALL but also in diverse human haematopoietic, mesenchymal and epithelial tumours. These results indicate that murine and human tumours experience common biological processes driven by orthologous genetic events in their malignant evolution. The highly concordant nature of genomic events encourages the use of genomically unstable murine cancer models in the discovery of biological driver events in the human oncogenome.
Subject(s)
Chromosomal Instability/genetics , Chromosome Aberrations , Conserved Sequence/genetics , Leukemia-Lymphoma, Adult T-Cell/genetics , Lymphoma, T-Cell/genetics , Animals , Genome/genetics , Humans , Mice , PTEN Phosphohydrolase/deficiency , PTEN Phosphohydrolase/genetics , Synteny/geneticsABSTRACT
Notch receptors transmit signals between adjacent cells. Signaling is initiated when ligand binding induces metalloprotease cleavage of Notch within an extracellular negative regulatory region (NRR). We present here the X-ray structure of the human NOTCH2 NRR, which adopts an autoinhibited conformation. Extensive interdomain interactions within the NRR bury the metalloprotease site, showing that a substantial conformational movement is necessary to expose this site during activation by ligand. Leukemia-associated mutations in NOTCH1 probably release autoinhibition by destabilizing the conserved hydrophobic core of the NRR.
Subject(s)
Receptor, Notch2/antagonists & inhibitors , Receptor, Notch2/chemistry , Crystallography, X-Ray , Genes, Reporter , Humans , Models, Molecular , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
Gain-of-function NOTCH1 mutations are found in 50%-70% of human T cell acute lymphoblastic leukemia/lymphoma (T-ALL) cases. Gain-of-function NOTCH1 alleles that initiate strong downstream signals induce leukemia in mice, but it is unknown whether the gain-of-function NOTCH1 mutations most commonly found in individuals with T-ALL generate downstream signals of sufficient strength to induce leukemia. We addressed this question by expressing human gain-of-function NOTCH1 alleles of varying strength in mouse hematopoietic precursors. Uncommon gain-of-function NOTCH1 alleles that initiated strong downstream signals drove ectopic T cell development and induced leukemia efficiently. In contrast, although gain-of-function alleles that initiated only weak downstream signals also induced ectopic T cell development, these more common alleles failed to efficiently initiate leukemia development. However, weak gain-of-function NOTCH1 alleles accelerated the onset of leukemia initiated by constitutively active K-ras and gave rise to tumors that were sensitive to Notch signaling pathway inhibition. These data show that induction of leukemia requires doses of Notch1 greater than those needed for T cell development and that most NOTCH1 mutations found in T-ALL cells do not generate signals of sufficient strength to initiate leukemia development. Furthermore, low, nonleukemogenic levels of Notch1 can complement other leukemogenic events, such as activation of K-ras. Even when Notch1 participates secondarily, the resulting tumors show "addiction" to Notch, providing a further rationale for evaluating Notch signaling pathway inhibitors in leukemia.
Subject(s)
Gene Expression Regulation, Leukemic , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Receptor, Notch1/genetics , Receptor, Notch1/physiology , Alleles , Animals , Cell Line, Tumor , Genes, ras , Humans , Mice , Mice, Inbred C57BL , Models, Biological , Models, Genetic , Mutation , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Signal Transduction , Time FactorsABSTRACT
The NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and gamma-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability. We show here that 15 of the 16 mutations activate canonical NOTCH1 signaling. Increases in signaling occur in a ligand-independent fashion, require gamma-secretase activity, and correlate with an increased susceptibility to cleavage by metalloproteases. The activating mutations cause soluble NOTCH1 heterodimers to dissociate more readily, either under native conditions (n = 3) or in the presence of urea (n = 11). One mutation, an insertion of 14 residues immediately N terminal to the metalloprotease cleavage site, increases metalloprotease sensitivity more than all others, despite a negligible effect on heterodimer stability by comparison, suggesting that the insertion may expose the S2 site by repositioning it relative to protective NOTCH1 ectodomain residues. Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.
Subject(s)
Leukemia-Lymphoma, Adult T-Cell/genetics , Leukemia-Lymphoma, Adult T-Cell/metabolism , Mutation , Receptor, Notch1/chemistry , Receptor, Notch1/genetics , Amino Acid Sequence , Binding Sites/genetics , Cell Line , Dimerization , Humans , Ligands , Models, Biological , Molecular Sequence Data , Protein Structure, Quaternary , Protein Structure, Tertiary , Receptor, Notch1/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
NOTCH1 is a large type I transmembrane receptor that regulates normal T-cell development via a signaling pathway that relies on regulated proteolysis. Ligand binding induces proteolytic cleavages in NOTCH1 that release its intracellular domain (ICN1), which translocates to the nucleus and activates target genes by forming a short-lived nuclear complex with two other proteins, the DNA-binding factor CSL and a Mastermind-like (MAML) coactivator. Recent work has shown that human T-ALL is frequently associated with C-terminal NOTCH1 truncations, which uniformly remove sequences lying between residues 2524 and 2556. This region includes the highly conserved sequence WSSSSP (S4), which based on its amino acid content appeared to be a likely site for regulatory serine phosphorylation events. We show here that the mutation of the S4 sequence leads to hypophosphorylation of ICN1; increased NOTCH1 signaling; and the stabilization of complexes containing ICN1, CSL, and MAML1. Consistent with these in vitro studies, mutation of the WSSSSP sequence converts nonleukemogenic weak gain-of-function NOTCH1 alleles into alleles that cause aggressive T-ALLs in a murine bone marrow transplant model. These studies indicate that S4 is an important negative regulatory sequence and that the deletion of S4 likely contributes to the development of human T-ALL.
Subject(s)
Conserved Sequence/genetics , Down-Regulation/genetics , Neoplasms/pathology , Receptor, Notch1/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Amino Acid Sequence , Animals , Cells, Cultured , Cyclin-Dependent Kinase 8 , Cyclin-Dependent Kinases/metabolism , Humans , Leukemia-Lymphoma, Adult T-Cell/pathology , Mice , Molecular Sequence Data , Mutation/genetics , NIH 3T3 Cells , Peptides/chemistry , Phosphorylation , Protein Structure, Tertiary , Proviruses/genetics , Receptor, Notch1/chemistry , ThermodynamicsABSTRACT
BACKGROUND: The functional interchangeability of mammalian Notch receptors (Notch1-4) in normal and pathophysiologic contexts such as cancer is unsettled. We used complementary in vivo, cell-based and structural analyses to compare the abilities of activated Notch1-4 to support T cell development, induce T cell acute lymphoblastic leukemia/lymphoma (T-ALL), and maintain T-ALL cell growth and survival. PRINCIPAL FINDINGS: We find that the activated intracellular domains of Notch1-4 (ICN1-4) all support T cell development in mice and thymic organ culture. However, unlike ICN1-3, ICN4 fails to induce T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) and is unable to rescue the growth of Notch1-dependent T-ALL cell lines. The ICN4 phenotype is mimicked by weak gain-of-function forms of Notch1, suggesting that it stems from a failure to transactivate one or more critical target genes above a necessary threshold. Experiments with chimeric receptors demonstrate that the Notch ankyrin repeat domains differ in their leukemogenic potential, and that this difference correlates with activation of Myc, a direct Notch target that has an important role in Notch-associated T-ALL. CONCLUSIONS/SIGNIFICANCE: We conclude that the leukemogenic potentials of Notch receptors vary, and that this functional difference stems in part from divergence among the highly conserved ankyrin repeats, which influence the transactivation of specific target genes involved in leukemogenesis.
Subject(s)
Ankyrin Repeat , Cell Transformation, Neoplastic/pathology , Genetic Variation , Proto-Oncogene Proteins c-myc/genetics , Receptors, Notch/chemistry , Receptors, Notch/metabolism , Transcriptional Activation/genetics , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Animals , Biophysical Phenomena/drug effects , Bone Marrow Transplantation , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Transformation, Neoplastic/drug effects , Genetic Variation/drug effects , Humans , Mice , Organ Culture Techniques , Peptides/chemistry , Peptides/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protease Inhibitors/pharmacology , Proto-Oncogene Proteins c-myc/metabolism , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Structure-Activity Relationship , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Thymocytes/drug effects , Thymocytes/metabolism , Thymocytes/pathology , Transcriptional Activation/drug effects , Transduction, GeneticABSTRACT
BACKGROUND: Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia. PRINCIPAL FINDINGS: The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity. CONCLUSIONS/SIGNIFICANCE: S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or T-ALL-associated mutations lead to conformational changes of the NRR that permit metalloprotease cleavage.
Subject(s)
Receptor, Notch1/metabolism , Receptor, Notch2/metabolism , Signal Transduction , Amino Acid Sequence , Dimerization , Furin/metabolism , Humans , Hydrolysis , Models, Molecular , Molecular Sequence Data , Mutation , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Receptor, Notch1/chemistry , Receptor, Notch1/genetics , Receptor, Notch2/chemistry , Receptor, Notch2/genetics , Sequence Homology, Amino Acid , X-Ray DiffractionABSTRACT
The Notch pathway regulates the development of many tissues and cell types and is involved in a variety of human diseases, making it an attractive potential therapeutic target. This promise has been limited by the absence of potent inhibitors or agonists that are specific for individual human Notch receptors (NOTCH1-4). Using an unbiased functional screening, we identified monoclonal antibodies that specifically inhibit or induce activating proteolytic cleavages in NOTCH3. Remarkably, the most potent inhibitory and activating antibodies bind to overlapping epitopes within a juxtamembrane negative regulatory region that protects NOTCH3 from proteolysis and activation in its resting autoinhibited state. The inhibitory antibodies revert phenotypes conveyed on 293T cells by NOTCH3 signaling, such as increased cellular proliferation, survival, and motility, whereas the activating antibody mimics some of the effects of ligand-induced Notch activation. These findings provide insights into the mechanisms of Notch autoinhibition and activation and pave the way for the further development of specific antibody-based modulators of the Notch receptors, which are likely to be of utility in a wide range of experimental and therapeutic settings.