ABSTRACT
BACKGROUND: Clinical trials of the KRAS inhibitors adagrasib and sotorasib have shown promising activity in cancers harboring KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRASG12C). The mechanisms of acquired resistance to these therapies are currently unknown. METHODS: Among patients with KRASG12C -mutant cancers treated with adagrasib monotherapy, we performed genomic and histologic analyses that compared pretreatment samples with those obtained after the development of resistance. Cell-based experiments were conducted to study mutations that confer resistance to KRASG12C inhibitors. RESULTS: A total of 38 patients were included in this study: 27 with non-small-cell lung cancer, 10 with colorectal cancer, and 1 with appendiceal cancer. Putative mechanisms of resistance to adagrasib were detected in 17 patients (45% of the cohort), of whom 7 (18% of the cohort) had multiple coincident mechanisms. Acquired KRAS alterations included G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, and high-level amplification of the KRASG12C allele. Acquired bypass mechanisms of resistance included MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3; and loss-of-function mutations in NF1 and PTEN. In two of nine patients with lung adenocarcinoma for whom paired tissue-biopsy samples were available, histologic transformation to squamous-cell carcinoma was observed without identification of any other resistance mechanisms. Using an in vitro deep mutational scanning screen, we systematically defined the landscape of KRAS mutations that confer resistance to KRASG12C inhibitors. CONCLUSIONS: Diverse genomic and histologic mechanisms impart resistance to covalent KRASG12C inhibitors, and new therapeutic strategies are required to delay and overcome this drug resistance in patients with cancer. (Funded by Mirati Therapeutics and others; ClinicalTrials.gov number, NCT03785249.).
Subject(s)
Acetonitriles/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Colorectal Neoplasms/drug therapy , Drug Resistance, Neoplasm/genetics , Lung Neoplasms/drug therapy , Mutation , Piperazines/therapeutic use , Proto-Oncogene Proteins p21(ras)/genetics , Pyrimidines/therapeutic use , Appendiceal Neoplasms/drug therapy , Appendiceal Neoplasms/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Colorectal Neoplasms/genetics , Humans , Lung Neoplasms/genetics , Protein Conformation , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/ultrastructure , Pyridines/therapeutic useABSTRACT
Cytotoxic T cells targeting cancer neoantigens harboring driver mutations can lead to durable tumor regression in an HLAI-dependent manner. However, it is difficult to extend the population of patients who are eligible for neoantigen-based immunotherapy, as immunogenic neoantigen-HLA pairs are rarely shared across different patients. Thus, a way to find other human leukocyte antigen (HLA) alleles that can also present a clinically effective neoantigen is needed. Recently, neoantigen-based immunotherapy targeting the KRAS G12D mutation in patients with HLA-C*08:02 has shown effectiveness. In a proof-of-concept study, we proposed a combinatorial strategy (the combination of phylogenetic and structural analyses) to find potential HLA alleles that could also present KRAS G12D neoantigen. Compared to in silico binding prediction, this strategy avoids the uneven accuracy across different HLA alleles. Our findings extend the population of patients who are potentially eligible for immunotherapy targeting the KRAS G12D mutation. Additionally, we provide an alternative way to predict neoantigen-HLA pairs, which maximizes the clinical usage of shared neoantigens.
Subject(s)
Antigens, Neoplasm/genetics , HLA-C Antigens/genetics , Mutation , Neoplasms/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Antigens, Neoplasm/immunology , Epitopes , HLA-C Antigens/metabolism , HLA-C Antigens/ultrastructure , Humans , Immunotherapy , Major Histocompatibility Complex , Neoplasms/immunology , Phylogeny , Protein Structure, Tertiary , Proto-Oncogene Proteins p21(ras)/metabolism , Proto-Oncogene Proteins p21(ras)/ultrastructureABSTRACT
Nav1.5, encoded by SCN5A, has been associated with metastasis in colorectal cancer (CRC). Here, we investigated the mechanism by which Nav1.5 regulates tumor progression and whether Nav1.5 influences chemosensitivity to 5-fluorouracil (5-FU) in CRCs. CRC cases were evaluated for Nav1.5 expression. Elevated Nav1.5 expression was associated with poor prognosis in CRCs, whereas stage II/III patients with upregulated SCN5A expression could have better survival after receiving 5-FU-based adjuvant chemotherapy. In CRC cells, SCN5A knockdown reduced the proliferation, migration and invasion. According to RNA sequencing, SCN5A knockdown inhibited both the cell cycle and epithelial-mesenchymal transition. In addition, Nav1.5 stabilized the KRas-calmodulin complex to modulate Ras signaling, promoting Ca2+ influx through the Na+-Ca2+ exchanger and Ca2+ release-activated calcium channel. Meanwhile, SCN5A knockdown increased the 50% inhibitory concentration to 5-FU by upregulating 5-FU-stimulated apoptosis in CRCs. In conclusion, Nav1.5 could progress to proliferation and metastasis through Ca2+/calmodulin-dependent Ras signaling in CRC, and it could also enhance 5-FU-stimulated apoptosis. Clinically, patients with stage II/III CRCs with elevated SCN5A expression demonstrated poor prognosis, yet those patients could benefit more from 5-FU-based chemotherapy than patients with lower SCN5A expression.
Subject(s)
Calmodulin/genetics , Colorectal Neoplasms/drug therapy , Fluorouracil/pharmacology , NAV1.5 Voltage-Gated Sodium Channel/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Apoptosis/drug effects , Calmodulin/ultrastructure , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Chemotherapy, Adjuvant/adverse effects , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Disease Progression , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Epithelial-Mesenchymal Transition/drug effects , Fluorouracil/adverse effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Multiprotein Complexes/genetics , Multiprotein Complexes/ultrastructure , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Proto-Oncogene Proteins p21(ras)/ultrastructureABSTRACT
Decades ago, Rap1, a small GTPase very similar to Ras, was observed to suppress oncogenic Ras phenotype, reverting its transformation. The proposed reason, persisting since, has been competition between Ras and Rap1 for a common target. Yet, none was found. There was also Rap1's puzzling suppression of Raf-1 versus activation of BRAF. Reemerging interest in Rap1 envisages capturing its Ras suppression action by inhibitors. Here, we review the literature and resolve the enigma. In vivo oncogenic Ras exists in isoform-distinct nanoclusters. The presence of Rap1 within the nanoclusters reduces the number of the clustered oncogenic Ras molecules, thus suppressing Raf-1 activation and mitogen-activated protein kinase (MAPK) signaling. Nanoclustering suggests that Rap1 suppression is Ras isoform dependent. Altogether, a potent Rap1-like inhibitor appears unlikely.
Subject(s)
Neoplasms/pathology , Proto-Oncogene Proteins c-raf/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , rap1 GTP-Binding Proteins/metabolism , Crystallography , Humans , MAP Kinase Signaling System , Models, Molecular , Protein Binding , Protein Domains , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins B-raf/ultrastructure , Proto-Oncogene Proteins c-raf/ultrastructure , Proto-Oncogene Proteins p21(ras)/ultrastructure , rap GTP-Binding Proteins/metabolism , rap1 GTP-Binding Proteins/ultrastructureABSTRACT
RAS regulation and signaling are largely accomplished by direct protein-protein interactions, making RAS protein dynamics a critical determinant of RAS function. Here, we report a crystal structure of GDP-bound KRASV14I, a mutated KRAS variant associated with the developmental RASopathy disorder Noonan syndrome (NS), at 1.5-1.6 Å resolution. The structure is notable for revealing a marked extension of switch 1 away from the G-domain and nucleotide-binding site of the KRAS protein. We found that this extension is associated with a loss of the magnesium ion and a tilt in the position of the guanine base because of the additional carbon introduced by the isoleucine substitution. Hydrogen-deuterium exchange MS analysis confirmed that this conformation occurs in solution, but also disclosed a difference in kinetics when compared with KRASA146T, another RAS mutant that displays a nearly identical conformation in previously reported crystal structures. This conformational change contributed to a high rate of guanine nucleotide-exchange factor (GEF)-dependent and -independent nucleotide exchange and to an increase in affinity for SOS Ras/Rac GEF 1 (SOS1), which appears to be the major mode of activation for this RAS variant. These results highlight a mechanistic connection between KRASA146T and KRASV14I that may have implications for the regulation of these variants and for the development of therapeutic strategies to manage KRAS variant-associated disorders.
Subject(s)
Proto-Oncogene Proteins p21(ras)/metabolism , Proto-Oncogene Proteins p21(ras)/ultrastructure , Binding Sites , Crystallography, X-Ray/methods , Enzyme Activation , GTP Phosphohydrolases/ultrastructure , Guanine Nucleotide Exchange Factors/metabolism , Humans , Kinetics , Models, Molecular , Noonan Syndrome/metabolism , Nucleotides/metabolism , Polymorphism, Single Nucleotide , Protein Conformation , Signal Transduction , Structure-Activity Relationship , ras Guanine Nucleotide Exchange Factors/metabolism , ras Proteins/genetics , ras Proteins/metabolismABSTRACT
KRas4b is a small G-protein whose constitutively active oncogenic mutants are present in 90% of pancreatic cancers. Using fully post-translationally modified KRAS4b, we investigated the role of lipid identity in the recruitment of KRas4b to a membrane surface of defined composition. Application of a newly developed single frequency fluorescence anisotropy decay experiment to this system revealed that KRas4b has a significant binding preference for Nanodisc bilayers containing PIP2. We conducted molecular dynamics simulations to look for an origin of this specificity. In the case of membranes containing PIP2 the protein formed long-lived salt bridges with PIP2 head groups but not the monovalent DMPS, explaining the experimentally observed lipid specificity. Additionally, we report that PIP2 forms key contacts with Helix-4 on the catalytic domain of KRas4b that orient the protein in a manner expected to facilitate association with upstream and downstream signaling partners.
Subject(s)
Anions/chemistry , Lipid Bilayers/chemistry , Molecular Docking Simulation , Phosphatidylinositol 4,5-Diphosphate/chemistry , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/ultrastructure , Binding Sites , Models, Chemical , Protein Binding , Protein Conformation , Structure-Activity RelationshipABSTRACT
In the last decade evidence has accumulated that small domains of 50-700 nm in diameter are located in the exoplasmic leaflet of the plasma membrane. Most of these domains supposedly consist of specific sets of lipids and proteins, and are believed to coordinate signal transduction cascades. Whether similar domains are also present in the cytoplasmic leaflet of the plasma membrane is unclear so far. To investigate the presence of cytoplasmic leaflet domains, the H-Ras membrane-targeting sequence was fused to the C-terminus of the enhanced yellow fluorescent protein. Using single-molecule fluorescence microscopy, trajectories of individual molecules diffusing in the cytoplasmic leaflet of the plasma membrane were recorded. From these trajectories, the diffusion of individual membrane-anchored enhanced yellow fluorescent protein molecules was studied in live cells on timescales from 5 to 200 ms. The results show that the diffusion of 30-40% of the molecules is constrained in domains with a typical size of 200 nm. Neither breakdown of actin nor cholesterol extraction changed the domain characteristics significantly, indicating that the observed domains may not be related to the membrane domains identified so far.
Subject(s)
Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cytoplasm/metabolism , Membrane Microdomains/metabolism , Membrane Microdomains/ultrastructure , Proto-Oncogene Proteins p21(ras)/metabolism , Proto-Oncogene Proteins p21(ras)/ultrastructure , 3T3 Cells , Animals , Cell Line , Humans , Kidney/cytology , Kidney/embryology , Kidney/metabolism , Mice , Protein Transport/physiology , Tissue DistributionABSTRACT
Previously, we demonstrated the interaction of homologous linear duplexes with formation of four-way DNA structures on the model of five PCR products. We propose that homologous duplex interaction is initiated by the nucleation of several dissociated base pairs of the complementary ends of two fragments with Holliday junction formation, in which cross point migration occurs via spooling of DNA strands from one duplex to the other one, finally resulting in complete resolution into new or previously existing duplexes. To confirm that DNA-DNA interaction involves formation of four-way DNA structures with strand exchange at the cross point, we have demonstrated the strand exchange process between identical duplexes using homologous fragments, harboring either biotin label or (32)P-label. Incubation of the mixture resulted in the addition of (32)P-label to biotin-labeled fragments, and the intensity of (32)P-labeling of biotinylated fragments was dependent upon the incubation duration. DNA-DNA interaction is not based on surface-dependent denaturing, as Triton X-100 does not decrease the formation of complexes between DNA duplexes. The equilibrium concentration of Holliday junctions depends on the sequences of the fragment ends and the incubation temperature. The free energy of Holliday junction formation by the fragments with GC and AT ends differed by 0.6 kcal/mol. Electron microscopic analysis demonstrated that the majority of Holliday junctions harbor the cross point within a 300 base pair region of the fragment ends. This insight into the mechanism of homologous duplex interaction extends our understanding of different DNA rearrangements. Understanding of DNA-DNA interaction is of practical use for better interpretation and optimization of PCR-based analyses.
Subject(s)
DNA/chemistry , Nucleic Acid Conformation , Nucleic Acid Heteroduplexes/chemistry , Sequence Homology, Nucleic Acid , DNA/ultrastructure , DNA Primers/chemistry , Humans , Microscopy, Electron , Nucleic Acid Heteroduplexes/ultrastructure , Polypropylenes , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/ultrastructure , Surface Properties , Thermodynamics , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/ultrastructureABSTRACT
We have employed ESR spectroscopy using guanine nucleotides that contain a spin label at the 2',3'-position of the ribose to investigate structural changes in the proto-oncogene product p21(ras) that are dependent on nucleotide hydrolysis. The three nucleotide analogs used were 2',3'-(2,2,5, 5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid ester (SL) GTP, SL-GDP, and the non-hydrolyzable analog SL-guanylylimidodiphosphate. SL-GTP was hydrolyzed by p21 with rates similar to those for GTP hydrolysis and appears to be an excellent substrate analog. The ESR spectra of SL-GTP and SL-GDP in complex with p21 differ significantly when acquired at 0 degrees C or 5 degrees C indicating different environments (conformations) of the protein-bound radicals depending on the phosphorylation state of the bound nucleotide. We calculated the rate constant for the conformational change as deduced from the changes in the corresponding ESR spectra upon incubation of the p21.SL-GTP complex at 25 degrees C and compared it to the rate constant of hydrolysis of SL-GTP at the same temperature. The rate constant deduced from the ESR method was similar to that determined by a high performance liquid chromatography technique. The data are in agreement with the idea that a conformational change during GTP hydrolysis by p21 occurs simultaneously with the actual hydrolysis step.
Subject(s)
Guanosine Diphosphate/analogs & derivatives , Guanosine Triphosphate/analogs & derivatives , Proto-Oncogene Proteins p21(ras)/ultrastructure , Binding Sites , Electron Spin Resonance Spectroscopy , Guanylyl Imidodiphosphate/analogs & derivatives , Hydrolysis , Kinetics , Magnesium/metabolism , Protein Structure, Tertiary , Recombinant Proteins , Spin Labels , TemperatureABSTRACT
Although Ras residue phenylalanine-156 (F156) is strictly conserved in all members of the Ras superfamily of proteins, it is located outside of the consensus GDP/GTP-binding pocket. Its location within the hydrophobic core of Ras suggests that its strict conservation reflects a crucial role in structural stability. However, mutation of the equivalent residue (F157L) in the Drosophila Ras-related protein Rap results in a gain-of-function phenotype, suggesting an alternative role for this residue. Therefore, we have introduced an F156L mutation into Ras to evaluate the role of this residue in Ras structure and function. Whereas introduction of this mutation activated the transforming potential of wild-type Ras, it did not impair that of oncogenic Ras. Further, Ras (156L) exhibited an extremely rapid off rate for bound GDP/GTP in vitro and showed increased levels of Ras.GTP in vivo. To determine the structural basis for these altered properties, we used high-resolution nuclear magnetic resonance spectroscopy. The F156L mutation caused loss of contact with residues 6, 23, 55, and 79, resulting in disruption of secondary structure in alpha-helix 1 and in beta-sheets 1-5. These major structural changes contrast with the isolated alterations induced by oncogenic mutation (residues 12 or 61) that perturb GTPase activity, and instead, weaken Ras contacts with Mg2+ and its guanine nucleotide substrate and result in increased rates of GDP/GTP dissociation. Altogether, these observations demonstrate the essential role of this conserved residue in Ras structure and its function as a regulated GDP/GTP switch.
Subject(s)
GTP-Binding Proteins/metabolism , Genes, ras , Proto-Oncogene Proteins p21(ras)/metabolism , 3T3 Cells , Animals , Cell Transformation, Neoplastic , GTP-Binding Proteins/ultrastructure , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Mutagenesis, Site-Directed , Phenylalanine , Protein Structure, Secondary , Protein Structure, Tertiary , Proto-Oncogene Proteins p21(ras)/ultrastructure , Structure-Activity RelationshipSubject(s)
GTP-Binding Proteins/chemistry , Transducin/chemistry , Animals , Cattle , Crystallography , GTP Phosphohydrolases/chemistry , GTP-Binding Proteins/ultrastructure , Guanosine Triphosphate/metabolism , Protein Conformation , Protein Structure, Secondary , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/ultrastructure , Transducin/ultrastructureABSTRACT
The biological functions of ras proteins are controlled by the bound guanine nucleotide GDP or GTP. The GTP-bound conformation is biologically active, and is rapidly deactivated to the GDP-bound conformation through interaction with GAP (GTPase Activating Protein). Most transforming mutants of ras proteins have drastically reduced GTP hydrolysis rates even in the presence of GAP. The crystal structures of the GDP complexes of ras proteins at 2.2 A resolution reveal the detailed interaction between the ras proteins and the GDP molecule. All the currently known transforming mutation positions are clustered around the bound guanine nucleotide molecule. The presumed "effector" region and the GAP recognition region are both highly exposed. No significant structural differences were found between the GDP complexes of normal ras protein and the oncogenic mutant with valine at position 12, except the side-chain of the valine residue. However, comparison with GTP-analog complexes of ras proteins suggests that the valine side-chain may inhibit GTP hydrolysis in two possible ways: (1) interacting directly with the gamma-phosphate and altering its orientation or the conformation of protein residues around the phosphates; and/or (2) preventing either the departure of gamma-phosphate on GTP hydrolysis or the entrance of a nucleophilic group to attack the gamma-phosphate. The structural similarity between ras protein and the bacterial elongation factor Tu suggests that their common structural motif might be conserved for other guanine nucleotide binding proteins.