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Current Opinion in Chemical Biology Jun 2021The protein KRAS has for decades been considered a holy grail of cancer drug discovery. For most of that time, it has also been considered undruggable. Since 2018, five... (Review)
Review
The protein KRAS has for decades been considered a holy grail of cancer drug discovery. For most of that time, it has also been considered undruggable. Since 2018, five compounds have entered the clinic targeting a single mutant form of KRAS, G12C. Here, we review each of these compounds along with additional approaches to targeting this and other mutants. Remaining challenges include expanding the identification of inhibitors to a broader range of known mutants and to conformations of the protein more likely to avoid development of resistance.
Topics: Acetonitriles; Animals; Antineoplastic Agents; Drug Design; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; Mutant Proteins; Mutation; Piperazines; Precision Medicine; Protein Binding; Protein Conformation; Proto-Oncogene Proteins p21(ras); Pyridines; Pyrimidines; Quinazolines; Structure-Activity Relationship
PubMed: 33838397
DOI: 10.1016/j.cbpa.2021.02.010 -
Trends in Genetics : TIG Aug 2022Gene disruption or knockout is an essential tool for elucidating gene function. Conditional knockout methodology was developed to further advance these studies by... (Review)
Review
Gene disruption or knockout is an essential tool for elucidating gene function. Conditional knockout methodology was developed to further advance these studies by enabling gene disruption at a predefined time and/or in discrete cells. While the conditional knockout method is widely used in the mouse, technical limitations have stifled direct adoption of this methodology in other animal models including the zebrafish. Recent advances in genome editing have enabled engineering of distinct classes of conditional mutants in zebrafish. To further accelerate the development and application of conditional mutants, we will review diverse methods of conditional knockout engineering and discuss the advantages of different conditional alleles.
Topics: Alleles; Animals; Gene Editing; Mice; Mutagenesis; Phenotype; Zebrafish
PubMed: 35662532
DOI: 10.1016/j.tig.2022.04.007 -
Theranostics 2021Mitochondrial dysfunction and oxidative stress are frequently observed in the early stages of Alzheimer's disease (AD). Studies have shown that presenilin-1 (PS1), the...
Mitochondrial dysfunction and oxidative stress are frequently observed in the early stages of Alzheimer's disease (AD). Studies have shown that presenilin-1 (PS1), the catalytic subunit of γ-secretase whose mutation is linked to familial AD (FAD), localizes to the mitochondrial membrane and regulates its homeostasis. Thus, we investigated how five mutations (A431E, E280A, H163R, M146V, and Δexon9) observed in FAD affect mitochondrial functions. We used H4 glioblastoma cell lines genetically engineered to inducibly express either the wild-type PS1 or one of the five PS1 mutants in order to examine mitochondrial morphology, dynamics, membrane potential, ATP production, mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), oxidative stress, and bioenergetics. Furthermore, we used brains of PS1M146V knock-in mice, 3xTg-AD mice, and human AD patients in order to investigate the role of PS1 in regulating MAMs formation. Each PS1 mutant exhibited slightly different mitochondrial dysfunction. Δexon9 mutant induced mitochondrial fragmentation while A431E, E280A, H163R, and M146V mutants increased MAMs formation. A431E, E280A, M146V, and Δexon9 mutants also induced mitochondrial ROS production. A431E mutant impaired both complex I and peroxidase activity while M146V mutant only impaired peroxidase activity. All PS1 mutants compromised mitochondrial membrane potential and cellular ATP levels were reduced by A431E, M146V, and Δexon9 mutants. Through comparative profiling of hippocampal gene expression in PS1M146V knock-in mice, we found that PS1M146V upregulates Atlastin 2 (ATL2) expression level, which increases ER-mitochondria contacts. Down-regulation of ATL2 after PS1 mutant induction rescued abnormally elevated ER-mitochondria interactions back to the normal level. Moreover, ATL2 expression levels were significantly elevated in the brains of 3xTg-AD mice and AD patients. Overall, our findings suggest that each of the five FAD-linked mutations has a deleterious effect on mitochondrial functions in a variety of ways. The adverse effects of PS1 mutations on mitochondria may contribute to MAMs formation and oxidative stress resulting in an accelerated age of disease onset in people harboring mutant PS1.
Topics: Adenosine Triphosphate; Alzheimer Disease; Animals; Cell Line, Tumor; Endoplasmic Reticulum; Gene Knock-In Techniques; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria; Mutation; Oxidative Stress; Presenilin-1
PubMed: 34522215
DOI: 10.7150/thno.59776 -
Frontiers in Plant Science 2020Gametophytic mutants share very small proportion of the total mutants generated by any mutagenic approach; even rarer are the fertilization-defective gametophytic... (Review)
Review
Gametophytic mutants share very small proportion of the total mutants generated by any mutagenic approach; even rarer are the fertilization-defective gametophytic mutants. They require an efficient and targeted strategy instead of 'brute force' screening approach. The classical gametophyte mutant screening method, mainly based on the segregation distortion, can distinguish gametophytic mutants from the others. However, the mutants pooled after the screening constitute both fertilization-defective and developmental-defective gametophytic mutants. Until recently, there has not been any straightforward way to screen the former from the latter. Additionally, most of the mutations affecting both gametes are lost during the screening process. The novel gametophyte screening approach tends to circumvent those shortcomings. This review discusses on the classical approach of gametophytic mutant screening and focuses on the novel approach on distinguishing fertilization-/developmental-defective gametophytic mutants (both male and female). It offers an empirical basis of screening such mutants by taking in the consideration of earlier studies on fertilization failure, initiation of seed coat formation, and fertilization recovery system in plants.
PubMed: 32714355
DOI: 10.3389/fpls.2020.00967 -
Nature Communications Oct 2023The literature about mutant invasion and fixation typically assumes populations to exist in isolation from their ecosystem. Yet, populations are part of ecological...
The literature about mutant invasion and fixation typically assumes populations to exist in isolation from their ecosystem. Yet, populations are part of ecological communities, and enemy-victim (e.g. predator-prey or pathogen-host) interactions are particularly common. We use spatially explicit, computational pathogen-host models (with wild-type and mutant hosts) to re-visit the established theory about mutant fixation, where the pathogen equally attacks both wild-type and mutant individuals. Mutant fitness is assumed to be unrelated to infection. We find that pathogen presence substantially weakens selection, increasing the fixation probability of disadvantageous mutants and decreasing it for advantageous mutants. The magnitude of the effect rises with the infection rate. This occurs because infection induces spatial structures, where mutant and wild-type individuals are mostly spatially separated. Thus, instead of mutant and wild-type individuals competing with each other, it is mutant and wild-type "patches" that compete, resulting in smaller fitness differences and weakened selection. This implies that the deleterious mutant burden in natural populations might be higher than expected from traditional theory.
Topics: Humans; Models, Biological; Ecosystem; Probability; Population Dynamics
PubMed: 37863909
DOI: 10.1038/s41467-023-41787-5 -
Cellular Signalling Jan 2020Vav1 is physiologically active as a GDP/GTP nucleotide exchange factor (GEF) in the hematopoietic system. Its wild-type form was recently implicated in mammalian... (Review)
Review
Vav1 is physiologically active as a GDP/GTP nucleotide exchange factor (GEF) in the hematopoietic system. Its wild-type form was recently implicated in mammalian malignancies of hematologic and non-hematologic tissue origins. Moreover, it was recently identified as a mutated gene in human cancers of various origins. In this review we focus on the functional activities of several of the Vav1 mutants analyzed for their tumorigenic properties. We also discuss the relationship of the tested biochemical properties of Vav1 mutants, E59K, D517E and L801P, to their computer-based predicted properties. These comparisons further enhance the need for integration of computation-based structural analyses with experimental data in order to fully appreciate the activity of mutant proteins. Our comprehensive evaluation supports the classification of Vav1 as a bona fide oncogene in human cancers.
Topics: Animals; Carcinogenesis; Humans; Mutant Proteins; Mutation; Neoplasms; Oncogenes; Proto-Oncogene Proteins c-vav
PubMed: 31654719
DOI: 10.1016/j.cellsig.2019.109438 -
Oncogene Feb 2022The tumor suppressive transcription factor p53 is frequently inactivated in cancer cells by missense mutations that cluster in the DNA binding domain. 30% hit mutational...
The tumor suppressive transcription factor p53 is frequently inactivated in cancer cells by missense mutations that cluster in the DNA binding domain. 30% hit mutational hotspot residues, resulting in a complete loss of transcriptional activity and mutant p53-driven chemotherapy resistance. Of the remaining 70% of non-hotspot mutants, many are partial loss-of-function (partial-LOF) mutants with residual transcriptional activity. The therapeutic consequences of a partial-LOF have remained largely elusive. Using a p53 mutation engineered to reduce DNA binding, we demonstrate that partial-LOF is sufficient to enhance oncogene-driven tumorigenesis in mouse models of lung and pancreatic ductal adenocarcinoma and acute myeloid leukemia. Interestingly, mouse and human tumors with partial-LOF mutations showed mutant p53 protein accumulation similar as known for hotspot mutants. Different from the chemotherapy resistance caused by p53-loss, the partial-LOF mutant sensitized to an apoptotic chemotherapy response and led to a survival benefit. Mechanistically, the pro-apoptotic transcriptional activity of mouse and human partial-LOF mutants was rescued at high mutant protein levels, suggesting that accumulation of partial-LOF mutants enables the observed apoptotic chemotherapy response. p53 non-hotspot mutants with partial-LOF, therefore, represent tumorigenic p53 mutations that need to be distinguished from other mutations because of their beneficial impact on survival in a therapy context.
Topics: Tumor Suppressor Protein p53
PubMed: 34907344
DOI: 10.1038/s41388-021-02141-5 -
Cancer Research Sep 2023Oncogenic point mutants of isocitrate dehydrogenases 1 and 2 (IDH2) generate 2-hydroxyglutarate, which inhibits lysine demethylases and increases heterochromatin. Tumor...
Oncogenic point mutants of isocitrate dehydrogenases 1 and 2 (IDH2) generate 2-hydroxyglutarate, which inhibits lysine demethylases and increases heterochromatin. Tumor cells expressing IDH mutants are sensitive to PARP inhibitors (PARPi), offering an opportunity to eliminate IDH-driven tumor cells in therapy. Expression of an oncogenic IDH1 mutant in cells leads to aberrant heterochromatin formation at DNA breaks and impairs DNA repair through homologous recombination (HR), providing a possible explanation for the PARPi sensitivity of IDH mutant cells. However, a recent study published in Molecular Cell shows that IDH mutant tumors do not display the genomic alterations associated with HR defects. Instead, IDH mutants induce heterochromatin-dependent DNA replication stress. Furthermore, PARP is activated by the replication stress induced by IDH mutants and required for suppressing the ensuing DNA damage, providing an alternative model to explain the susceptibility of IDH mutant cells to PARPis. This study presents a new example of oncogene-induced and heterochromatin-dependent replication stress, and a role of PARP in the response to the stress, extending the molecular basis for PARP-targeted therapy.
Topics: Humans; Poly(ADP-ribose) Polymerase Inhibitors; Heterochromatin; Mutation; DNA Damage; Neoplasms; Isocitrate Dehydrogenase
PubMed: 37433029
DOI: 10.1158/0008-5472.CAN-23-2015 -
Journal of Evolutionary Biology Feb 2023Mutant dynamics in fragmented populations have been studied extensively in evolutionary biology. Yet, open questions remain, both experimentally and theoretically. Some...
Mutant dynamics in fragmented populations have been studied extensively in evolutionary biology. Yet, open questions remain, both experimentally and theoretically. Some of the fundamental properties predicted by models still need to be addressed experimentally. We contribute to this by using a combination of experiments and theory to investigate the role of migration in mutant distribution. In the case of neutral mutants, while the mean frequency of mutants is not influenced by migration, the probability distribution is. To address this empirically, we performed in vitro experiments, where mixtures of GFP-labelled ("mutant") and non-labelled ("wid-type") murine cells were grown in wells (demes), and migration was mimicked via cell transfer from well to well. In the presence of migration, we observed a change in the skewedness of the distribution of the mutant frequencies in the wells, consistent with previous and our own model predictions. In the presence of de novo mutant production, we used modelling to investigate the level at which disadvantageous mutants are predicted to exist, which has implications for the adaptive potential of the population in case of an environmental change. In panmictic populations, disadvantageous mutants can persist around a steady state, determined by the rate of mutant production and the selective disadvantage (selection-mutation balance). In a fragmented system that consists of demes connected by migration, a steady-state persistence of disadvantageous mutants is also observed, which, however, is fundamentally different from the mutation-selection balance and characterized by higher mutant levels. The increase in mutant frequencies above the selection-mutation balance can be maintained in small ( ) demes as long as the migration rate is sufficiently small. The migration rate above which the mutants approach the selection-mutation balance decays exponentially with . The observed increase in the mutant numbers is not explained by the change in the effective population size. Implications for evolutionary processes in diseases are discussed, where the pre-existence of disadvantageous drug-resistant mutant cells or pathogens drives the response of the disease to treatments.
Topics: Animals; Mice; Selection, Genetic; Models, Genetic; Mutation; Population Dynamics; Biological Evolution
PubMed: 36514852
DOI: 10.1111/jeb.14131 -
Methods in Enzymology 2020Directed evolution has emerged as the most productive enzyme engineering method, with stereoselectivity playing a crucial role when evolving mutants for application in...
Directed evolution has emerged as the most productive enzyme engineering method, with stereoselectivity playing a crucial role when evolving mutants for application in synthetic organic chemistry and biotechnology. In order to reduce the screening effort (bottleneck of directed evolution), improved methods for the creation of small and smart mutant libraries have been developed, including the combinatorial active-site saturation test (CAST) which involves saturation mutagenesis at appropriate residues surrounding the binding pocket, and iterative saturation mutagenesis (ISM). Nevertheless, even CAST/ISM mutant libraries require a formidable screening effort. Thus far, rational design as the alternative protein engineering technique has had only limited success when aiming for stereoselectivity. Here, we highlight a recent methodology dubbed focused rational iterative site-specific mutagenesis (FRISM), in which mutant libraries are not involved. It makes use of the tools that were previously employed in traditional rational enzyme design, but, inspired by CAST/ISM, the process is performed in an iterative manner. Only a few predicted mutants need to be screened, a fast process which leads to the identification of highly enantioselective and sufficiently active mutants.
Topics: Directed Molecular Evolution; Mutagenesis; Mutagenesis, Site-Directed; Protein Engineering
PubMed: 32896283
DOI: 10.1016/bs.mie.2020.04.055