-
Nature Nov 2019Accumulation of mutant proteins is a major cause of many diseases (collectively called proteopathies), and lowering the level of these proteins can be useful for...
Accumulation of mutant proteins is a major cause of many diseases (collectively called proteopathies), and lowering the level of these proteins can be useful for treatment of these diseases. We hypothesized that compounds that interact with both the autophagosome protein microtubule-associated protein 1A/1B light chain 3 (LC3) and the disease-causing protein may target the latter for autophagic clearance. Mutant huntingtin protein (mHTT) contains an expanded polyglutamine (polyQ) tract and causes Huntington's disease, an incurable neurodegenerative disorder. Here, using small-molecule-microarray-based screening, we identified four compounds that interact with both LC3 and mHTT, but not with the wild-type HTT protein. Some of these compounds targeted mHTT to autophagosomes, reduced mHTT levels in an allele-selective manner, and rescued disease-relevant phenotypes in cells and in vivo in fly and mouse models of Huntington's disease. We further show that these compounds interact with the expanded polyQ stretch and could lower the level of mutant ataxin-3 (ATXN3), another disease-causing protein with an expanded polyQ tract. This study presents candidate compounds for lowering mHTT and potentially other disease-causing proteins with polyQ expansions, demonstrating the concept of lowering levels of disease-causing proteins using autophagosome-tethering compounds.
Topics: Alleles; Animals; Ataxin-3; Autophagosomes; Autophagy; Disease Models, Animal; Drosophila Proteins; Drosophila melanogaster; Drug Evaluation, Preclinical; Female; Humans; Huntingtin Protein; Male; Mice; Microtubule-Associated Proteins; Mutant Proteins; Mutation; Neurons; Peptides; Phenotype; Reproducibility of Results
PubMed: 31666698
DOI: 10.1038/s41586-019-1722-1 -
Cell Death and Differentiation May 2022Mutations in the TP53 tumour suppressor gene are found in ~50% of human cancers [1-6]. TP53 functions as a transcription factor that directly regulates the expression... (Review)
Review
Mutations in the TP53 tumour suppressor gene are found in ~50% of human cancers [1-6]. TP53 functions as a transcription factor that directly regulates the expression of ~500 genes, some of them involved in cell cycle arrest/cell senescence, apoptotic cell death or DNA damage repair, i.e. the cellular responses that together prevent tumorigenesis [1-6]. Defects in TP53 function not only cause tumour development but also impair the response of malignant cells to anti-cancer drugs, particularly those that induce DNA damage [1-6]. Most mutations in TP53 in human cancers cause a single amino acid substitution, usually within the DNA binding domain of the TP53 protein. These mutant TP53 proteins are often expressed at high levels in the malignant cells. Three cancer causing attributes have been postulated for mutant TP53 proteins: the inability to activate target genes controlled by wt TP53 (loss-of-function, LOF) that are critical for tumour suppression, dominant negative effects (DNE), i.e. blocking the function of wt TP53 in cells during early stages of transformation when mutant and wt TP53 proteins are co-expressed, and gain-of-function (GOF) effects whereby mutant TP53 impacts diverse cellular pathways by interacting with proteins that are not normally engaged by wt TP53 [1-6]. The GOF effects of mutant TP53 were reported to be essential for the sustained proliferation and survival of malignant cells and it was therefore proposed that agents that can remove mutant TP53 protein would have substantial therapeutic impact [7-9]. In this review article we discuss evidence for and against the value of targeting mutant TP53 protein for cancer therapy.
Topics: Antineoplastic Agents; Cell Transformation, Neoplastic; Humans; Mutant Proteins; Mutation; Neoplasms; Tumor Suppressor Protein p53
PubMed: 35332311
DOI: 10.1038/s41418-022-00962-9 -
Cell Death and Differentiation May 2022Mutation of the TP53 tumor suppressor gene is the most common genetic alteration in cancer, and almost 1000 alleles have been identified in human tumors. While virtually... (Review)
Review
Mutation of the TP53 tumor suppressor gene is the most common genetic alteration in cancer, and almost 1000 alleles have been identified in human tumors. While virtually all TP53 mutations are thought to compromise wild type p53 activity, the prevalence and recurrence of missense TP53 alleles has motivated countless research studies aimed at understanding the function of the resulting mutant p53 protein. The data from these studies support three distinct, but perhaps not necessarily mutually exclusive, mechanisms for how different p53 mutants impact cancer: first, they lose the ability to execute wild type p53 functions to varying degrees; second, they act as a dominant negative (DN) inhibitor of wild type p53 tumor-suppressive programs; and third, they may gain oncogenic functions that go beyond mere p53 inactivation. Of these possibilities, the gain of function (GOF) hypothesis is the most controversial, in part due to the dizzying array of biological functions that have been attributed to different mutant p53 proteins. Herein we discuss the current state of understanding of TP53 allele variation in cancer and recent reports that both support and challenge the p53 GOF model. In these studies and others, researchers are turning to more systematic approaches to profile TP53 mutations, which may ultimately determine once and for all how different TP53 mutations act as cancer drivers and whether tumors harboring distinct mutations are phenotypically unique. From a clinical perspective, such information could lead to new therapeutic approaches targeting the effects of different TP53 alleles and/or better sub-stratification of patients harboring TP53 mutant cancers.
Topics: Alleles; Carcinogenesis; Humans; Mutant Proteins; Mutation; Neoplasms; Tumor Suppressor Protein p53
PubMed: 35361963
DOI: 10.1038/s41418-022-00989-y -
Science Immunology Mar 2021Mutations in the oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are...
Mutations in the oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these -derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the and genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.
Topics: Amino Acid Sequence; Animals; Antibodies, Bispecific; Antigens, Neoplasm; Biomarkers, Tumor; Cell Line; Cross Reactions; HLA Antigens; Humans; Lymphocyte Activation; Mutant Proteins; Mutation; Peptide Fragments; Protein Binding; T-Lymphocyte Subsets; ras Proteins
PubMed: 33649101
DOI: 10.1126/sciimmunol.abd5515 -
Nature Sep 2021Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients. Both the number of...
Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients. Both the number of transferred T cells and their differentiation state are critical determinants of effective responses. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells and lower therapeutic efficacy, whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial. Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8 T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatment sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8 T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mouse models of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategy for creating new molecules with translational potential.
Topics: Animals; CD8-Positive T-Lymphocytes; Cell Differentiation; Drug Partial Agonism; Interleukin-2; Melanoma; Mice; Mitochondria; Mutant Proteins; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Receptors, Antigen, T-Cell; STAT5 Transcription Factor; Stem Cells; T Cell Transcription Factor 1; Translational Research, Biomedical
PubMed: 34526724
DOI: 10.1038/s41586-021-03861-0 -
Nature Nov 2023The skin epidermis is constantly renewed throughout life. Disruption of the balance between renewal and differentiation can lead to uncontrolled growth and tumour...
The skin epidermis is constantly renewed throughout life. Disruption of the balance between renewal and differentiation can lead to uncontrolled growth and tumour initiation. However, the ways in which oncogenic mutations affect the balance between renewal and differentiation and lead to clonal expansion, cell competition, tissue colonization and tumour development are unknown. Here, through multidisciplinary approaches that combine in vivo clonal analysis using intravital microscopy, single-cell analysis and functional analysis, we show how SmoM2-a constitutively active oncogenic mutant version of Smoothened (SMO) that induces the development of basal cell carcinoma-affects clonal competition and tumour initiation in real time. We found that expressing SmoM2 in the ear epidermis of mice induced clonal expansion together with tumour initiation and invasion. By contrast, expressing SmoM2 in the back-skin epidermis led to a clonal expansion that induced lateral cell competition without dermal invasion and tumour formation. Single-cell analysis showed that oncogene expression was associated with a cellular reprogramming of adult interfollicular cells into an embryonic hair follicle progenitor (EHFP) state in the ear but not in the back skin. Comparisons between the ear and the back skin revealed that the dermis has a very different composition in these two skin types, with increased stiffness and a denser collagen I network in the back skin. Decreasing the expression of collagen I in the back skin through treatment with collagenase, chronic UV exposure or natural ageing overcame the natural resistance of back-skin basal cells to undergoing EHFP reprogramming and tumour initiation after SmoM2 expression. Altogether, our study shows that the composition of the extracellular matrix regulates how susceptible different regions of the body are to tumour initiation and invasion.
Topics: Animals; Mice; Cell Transformation, Neoplastic; Collagen; Epidermis; Extracellular Matrix; Skin Neoplasms; Tumor Microenvironment; Carcinoma, Basal Cell; Ear; Collagenases; Aging; Ultraviolet Rays; Mutant Proteins
PubMed: 37968399
DOI: 10.1038/s41586-023-06740-y -
Cell Jan 2021Proton-coupled monocarboxylate transporters MCT1-4 catalyze the transmembrane movement of metabolically essential monocarboxylates and have been targeted for cancer...
Proton-coupled monocarboxylate transporters MCT1-4 catalyze the transmembrane movement of metabolically essential monocarboxylates and have been targeted for cancer treatment because of their enhanced expression in various tumors. Here, we report five cryo-EM structures, at resolutions of 3.0-3.3 Å, of human MCT1 bound to lactate or inhibitors in the presence of Basigin-2, a single transmembrane segment (TM)-containing chaperon. MCT1 exhibits similar outward-open conformations when complexed with lactate or the inhibitors BAY-8002 and AZD3965. In the presence of the inhibitor 7ACC2 or with the neutralization of the proton-coupling residue Asp309 by Asn, similar inward-open structures were captured. Complemented by structural-guided biochemical analyses, our studies reveal the substrate binding and transport mechanism of MCTs, elucidate the mode of action of three anti-cancer drug candidates, and identify the determinants for subtype-specific sensitivities to AZD3965 by MCT1 and MCT4. These findings lay out an important framework for structure-guided drug discovery targeting MCTs.
Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Basigin; Binding Sites; Cryoelectron Microscopy; Humans; Ligands; Models, Molecular; Monocarboxylic Acid Transporters; Mutant Proteins; Protons; Pyrimidinones; Rats; Structural Homology, Protein; Substrate Specificity; Symporters; Thiophenes
PubMed: 33333023
DOI: 10.1016/j.cell.2020.11.043 -
Nature Jan 2021Hutchinson-Gilford progeria syndrome (HGPS or progeria) is typically caused by a dominant-negative C•G-to-T•A mutation (c.1824 C>T; p.G608G) in LMNA, the gene that...
Hutchinson-Gilford progeria syndrome (HGPS or progeria) is typically caused by a dominant-negative C•G-to-T•A mutation (c.1824 C>T; p.G608G) in LMNA, the gene that encodes nuclear lamin A. This mutation causes RNA mis-splicing that produces progerin, a toxic protein that induces rapid ageing and shortens the lifespan of children with progeria to approximately 14 years. Adenine base editors (ABEs) convert targeted A•T base pairs to G•C base pairs with minimal by-products and without requiring double-strand DNA breaks or donor DNA templates. Here we describe the use of an ABE to directly correct the pathogenic HGPS mutation in cultured fibroblasts derived from children with progeria and in a mouse model of HGPS. Lentiviral delivery of the ABE to fibroblasts from children with HGPS resulted in 87-91% correction of the pathogenic allele, mitigation of RNA mis-splicing, reduced levels of progerin and correction of nuclear abnormalities. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. In transgenic mice that are homozygous for the human LMNA c.1824 C>T allele, a single retro-orbital injection of adeno-associated virus 9 (AAV9) encoding the ABE resulted in substantial, durable correction of the pathogenic mutation (around 20-60% across various organs six months after injection), restoration of normal RNA splicing and reduction of progerin protein levels. In vivo base editing rescued the vascular pathology of the mice, preserving vascular smooth muscle cell counts and preventing adventitial fibrosis. A single injection of ABE-expressing AAV9 at postnatal day 14 improved vitality and greatly extended the median lifespan of the mice from 215 to 510 days. These findings demonstrate the potential of in vivo base editing as a possible treatment for HGPS and other genetic diseases by directly correcting their root cause.
Topics: Adenine; Alleles; Alternative Splicing; Animals; Aorta; Base Pairing; Child; DNA; Disease Models, Animal; Female; Fibroblasts; Gene Editing; Humans; Lamin Type A; Longevity; Male; Mice; Mice, Transgenic; Mutant Proteins; Mutation; Progeria; RNA
PubMed: 33408413
DOI: 10.1038/s41586-020-03086-7 -
The Journal of Experimental Medicine Mar 2022Accumulation of tau has been implicated in various neurodegenerative diseases termed tauopathies. Tau is a microtubule-associated protein but is also actively released...
Accumulation of tau has been implicated in various neurodegenerative diseases termed tauopathies. Tau is a microtubule-associated protein but is also actively released into the extracellular fluids including brain interstitial fluid and cerebrospinal fluid (CSF). However, it remains elusive whether clearance of extracellular tau impacts tau-associated neurodegeneration. Here, we show that aquaporin-4 (AQP4), a major driver of the glymphatic clearance system, facilitates the elimination of extracellular tau from the brain to CSF and subsequently to deep cervical lymph nodes. Strikingly, deletion of AQP4 not only elevated tau in CSF but also markedly exacerbated phosphorylated tau deposition and the associated neurodegeneration in the brains of transgenic mice expressing P301S mutant tau. The current study identified the clearance pathway of extracellular tau in the central nervous system, suggesting that glymphatic clearance of extracellular tau is a novel regulatory mechanism whose impairment contributes to tau aggregation and neurodegeneration.
Topics: Animals; Aquaporin 4; Brain; Extracellular Fluid; Female; Glymphatic System; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mutant Proteins; Nerve Degeneration; Protein Aggregation, Pathological; tau Proteins
PubMed: 35212707
DOI: 10.1084/jem.20211275 -
Cell Feb 2021Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter...
Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter binding. Neurotransmitters that produce maximum open channel probabilities (Po) are full agonists, whereas those that yield lower than maximum Po are partial agonists. Cys-loop receptors are an important class of neurotransmitter receptors, yet a structure-based understanding of the mechanism of partial agonist action has proven elusive. Here, we study the glycine receptor with the full agonist glycine and the partial agonists taurine and γ-amino butyric acid (GABA). We use electrophysiology to show how partial agonists populate agonist-bound, closed channel states and cryo-EM reconstructions to illuminate the structures of intermediate, pre-open states, providing insights into previously unseen conformational states along the receptor reaction pathway. We further correlate agonist-induced conformational changes to Po across members of the receptor family, providing a hypothetical mechanism for partial and full agonist action at Cys-loop receptors.
Topics: Animals; Binding Sites; Cell Line; Cryoelectron Microscopy; Glycine; HEK293 Cells; Humans; Imaging, Three-Dimensional; Ion Channel Gating; Maleates; Models, Molecular; Mutant Proteins; Mutation; Neurotransmitter Agents; Protein Domains; Receptors, Glycine; Styrene; Zebrafish; gamma-Aminobutyric Acid
PubMed: 33567265
DOI: 10.1016/j.cell.2021.01.026