KRAS Protein Research Articles

Homonuclear Super-Resolution NMR Spectroscopy.

Homonuclear 1H NMR (nuclear magnetic resonance) spectra such as the [1H,1H]-NOESY (Nuclear Overhauser Enhancement spectroscopy) are essential for biomolecular structural biology. However, the spectrum contains hundreds to thousands of cross peaks meaning that within approximately 100 ppm2 there is significant signal overlap. Spectral resolution is thus a limiting factor for data interpretation for dynamics and structure elucidation. Acquiring the spectra at higher magnetic fields such as at a 1.2 GHz 1H frequency helps to reduce spectral crowding, since resolution scales proportionally to the magnetic field strength. Here, we show that the linewidths' of cross peaks in [1H,1H]-NOESY and [1H,1H]-TOCSY spectra can be further reduced by a factor of 2-3 in each dimension by super-resolution spectroscopy. In the indirect dimension a composite exponential-cosine weighted number of scans along the time increments are recorded and digitally smoothened by a window function. Furthermore, measurement time saving by reduced-acquisition super-resolution (RASR) is introduced. Application to the 20 kDa protein KRAS shows that highly resolved NMR spectra suitable for automated analysis can be acquired within less than 3 hours. The method opens an avenue towards automated chemical shift assignment, dynamics and structure determination of unlabeled small and medium size proteins within 24 hours.

Targeted protein degradation using chimeric human E2 ubiquitin-conjugating enzymes

Proteins can be targeted for degradation by engineering biomolecules that direct them to the eukaryotic ubiquitination machinery. For instance, the fusion of an E3 ubiquitin ligase to a suitable target binding domain creates a ‘biological Proteolysis-Targeting Chimera’ (bioPROTAC). Here we employ an analogous approach where the target protein is recruited directly to a human E2 ubiquitin-conjugating enzyme via an attached target binding domain. Through rational design and screening we develop E2 bioPROTACs that induce the degradation of the human intracellular proteins SHP2 and KRAS. Using global proteomics, we characterise the target-specific and wider effects of E2 vs. VHL-based fusions. Taking SHP2 as a model target, we also employ a route to bioPROTAC discovery based on protein display libraries, yielding a degrader with comparatively weak affinity capable of suppressing SHP2-mediated signalling.

Beyond KRAS(G12C): Biochemical and Computational Characterization of Sotorasib and Adagrasib Binding Specificity and the Critical Role of H95 and Y96.

Mutated KRAS proteins are frequently expressed in some of the most lethal human cancers and thus have been a target of intensive drug discovery efforts for decades. Lately, KRAS(G12C) switch-II pocket (SII-P)-targeting covalent small molecule inhibitors have finally reached clinical practice. Sotorasib (AMG-510) was the first FDA-approved covalent inhibitor to treat KRAS(G12C)-positive nonsmall cell lung cancer (NSCLC), followed soon by adagrasib (MRTX849). Both drugs target the GDP-bound state of KRAS(G12C), exploiting the strong nucleophilicity of acquired cysteine. Here, we evaluate the similarities and differences between sotorasib and adagrasib in their RAS SII-P binding by applying biochemical, cellular, and computational methods. Exact knowledge of SII-P engagement can enable targeting this site by reversible inhibitors for KRAS mutants beyond G12C. We show that adagrasib is strictly KRAS- but not KRAS(G12C)-specific due to its strong and unreplaceable interaction with H95. Unlike adagrasib, sotorasib is less dependent on H95 for its binding, making it a RAS isoform-agnostic compound, having a similar functionality also with NRAS and HRAS G12C mutants. Our results emphasize the accessibility of SII-P beyond oncogenic G12C and aid in understanding the molecular mechanism behind the clinically observed drug resistance, associated especially with secondary mutations on KRAS H95 and Y96.

Insight of the molecular mechanism of inhibitors located at different allosteric sites regulating the activity of wild type and mutant KRAS (G12)

As the important hub of many cellular signaling networks, KRAS (Kirsten rat sarcoma viral oncogene homologue) has been identified as a tumor biomarker. It is the frequently mutated oncogene in human cancers, and KRAS protein activation caused by mutations, such as G12D, has been found in many human tumors tissues. Although, there are two specific allosteric sites (AS1 and AS2) on the KRAS protein that can be used as the targets for inhibitor development, the difference of regulatory mechanisms between two individual allosteric sites still not be reported. Here, using molecular dynamics simulations combined with molecular mechanics generalized born surface area (MM/GBSA) analysis, we found that both of the inhibitors, located at AS1 and AS2, were able to reduce the binding free energy between wild type, mutant KRAS (G12/D/V/S/C) and GTP remarkably, however the effect of inhibitors on the binding free energy between wild type, mutant KRAS and GDP was limited. In addition, the degree of decrease of binding free energy between KRAS and GTP caused by inhibitors at AS2 was significantly greater than that caused by inhibitors at AS1. Further analysis revealed that both inhibitors at AS1 and AS2 were able to regulate the fluctuation of Switch Ⅰ and Switch Ⅱ to expand the pocket of the orthosteric site (GTP binding site), thereby reducing the binding of KRAS to GTP. Noteworthy there was significant differences in the regulatory preferences on Switch Ⅰ and Switch Ⅱ between two type inhibitor. The inhibitor at AS2 mainly regulated Switch Ⅱ to affect the pocket of the orthosteric site, while the inhibitor at AS1 mainly expand the pocket of the orthosteric site by regulating the fluctuation of Switch Ⅰ. Our study compared the differences between two type inhibitors in regulating the KRAS protein activity and revealed the advantages of the AS2 as the small molecule drug target, aiming to provide theoretical guidance for the research of novel KRAS protein inhibitors.

Targeted degradation of KRAS protein in non-small cell lung cancer: Therapeutic strategies using liposomal PROTACs with enhanced cellular uptake and pharmacokinetic profiles.

The role of KRAS mutation in non-small cell lung cancer (NSCLC) initiation and progression is well-established. However, "undruggable" KRAS protein poses the research of small molecule inhibitors a significant challenge. Addressing this, proteolysis-targeting chimeras (PROTACs) have become a cutting-edge treatment method, emphasizing protein degradation. A modified ethanol injection method was employed in this study to formulate liposomes encapsulating PROTAC drug LC-2 (LC-2 LPs). Precise surface modifications using cell-penetrating peptide R8 yielded R8-LC-2 liposomes (R8-LC-2 LPs). Comprehensive cellular uptake and cytotoxicity studies unveiled that R8-LC-2 LPs depended on concentration and time, showcasing the superior performance of R8-LC-2 LPs compared to normal liposomes. In vivo pharmacokinetic profiles demonstrated the capacity of DSPE-PEG2000 to prolong the circulation time of LC-2, leading to higher plasma concentrations compared to free LC-2. In vivo antitumor efficacy research underscored the remarkable ability of R8-LC-2 LPs to effectively suppress tumor growth. This study contributed to the exploration of enhanced therapeutic strategies for NSCLC, specifically focusing on the development of liposomal PROTACs targeting the "undruggable" KRAS protein. The findings provide valuable insights into the potential of this innovative approach, offering prospects for improved drug delivery and heightened antitumor efficacy.

Insight into structural dynamics involved in activation mechanism of full length KRAS wild type and P-loop mutants

KRAS protein is known to be frequently mutated in various cancers. The most common mutations being at position 12, 13 and 61. The positions 12 and 13 form part of the phosphate binding region (P-loop) of KRAS. Owing to mutation, the protein remains in continuous active state and affects the normal cellular process. Understanding the structural changes owing to mutations in GDP-bound (inactive state) and GTP-bound (active state) may help in the design of better therapeutics. To understand the structural flexibility due to the mutations specifically located at P-loop regions (G12D, G12V and G13D), extensive molecular dynamics simulations (24 μs) have been carried for both inactive (GDP-bound) and active (GTP-bound) structures for the wild type and these mutants. The study revealed that the local structural changes at the site of mutations allosterically guide changes in distant regions of the protein through hydrogen bond and hydrophobic signalling network. The dynamic cross correlation analysis and the comparison of the correlated motions among different systems manifested that changes in SW-I, SW-II, α3 and the loop preceding α3 affects the interactions of GDP/GTP with different regions of the protein thereby affecting its hydrolysis. Further, the Markov state modelling analysis confirmed that the mutations, especially G13D imparts rigidity to structure compared to wild type and thus limiting its conformational state in either intermediate state or active state. The study suggests that along with SW-I and SW-II regions, the loop region preceding the α3 helix and α3 helix are also involved in affecting the hydrolysis of nucleotides and may be considered while designing therapeutics against KRAS.

Potential of Oyster Mushroom (Pleurotus ostreatus) secondary metabolites on KRAS protein in-silico liver cancer

Liver Cell Carcinoma (KSH) currently ranks as the fourth most common cause of death due to cancer worldwide and is the sixth most common cancer in the world. HCC (Hepatocellular Carcinoma) is caused by environmental factors, viral infections, and certain genetic changes. Ras is a very important oncogene and is hypermutated in various types of cancer, including HCC. The doxorubicin compound is a curative agent that is widely used for chemotherapy treatment of various types of cancer, however, administration of the kura agent with high cytotoxic activity can cause several side effects. Secondary metabolite compounds have anti-oxidant capabilities including breaking free radical chains and chelating redox-active metal ions which cause lipid peroxidation. These properties also help prevent cancer. Pleurotus otstreatus bioactive compounds that have antioxidant potential, flavonol. Due to the potential cytotoxic activity of these secondary metabolites, a bioinformatics approach is needed in this research to determine the results of predicting the interaction activity of flavonol compounds on the KRAS protein in silico and to determine the amino acids and bonds involved in the interaction. between these compounds and the KRAS protein, as a reference for future HCC drugs. The method used is using the PyRx device, Biovia Discovery Studio, Autodock for the docking process and validating the docking results. The results of this research are that flavonol compounds have a docking value of -7.1 kcal/mol. This compound has the potential as a candidate compound that can be used in liver cancer therapy, because it’s have high values ​​and close to the positive control value, namely doxorubicin, of -7.6 kcal/mol.

Erlotinib Treatment in Colorectal Cancer Suppresses Autophagy Based on KRAS Mutation.

The KRAS gene is mutated in approximately 45% of colorectal cancer patients. There are currently very few targeted treatments or therapies equipped to directly inhibit KRAS due to its unusual structural intricacies. Erlotinib, an EGFR inhibitor, has previously been demonstrated to reduce cell viability by inducing autophagy in lung cancer cell lines with varying EGFR mutations. In contrast to lung cancer cells, evidence is provided herein for the first time that erlotinib treatment in colorectal cancer (CRC) cell lines reduces autophagy and still results in decreased cell viability. However, the effects of erlotinib in CRC cell lines containing a wildtype KRAS gene were different than in cells carrying a mutant KRAS gene. We show that there is significantly more downregulation of autophagy in KRAS mutant CRC cells compared to KRAS wildtype cells, both at transcriptional and translational levels, suggesting that the KRAS mutation is advantageous for cancer growth, even in the presence of erlotinib. Cell viability results determined that KRAS wildtype CRC cells had significantly more cell death compared to KRAS mutant cells. Using patient mRNA datasets, we showed that there was a significant correlation between the presence of the KRAS mutation and the expression of autophagy proteins. Additionally, through molecular dynamics simulations, we develop a blueprint for KRAS and autophagy protein interaction and the impact of the KRAS mutation on autophagy protein regulation. Overall, this is the first report of erlotinib treatment in CRC cells that assesses autophagy, and we demonstrate that autophagy activity is downregulated in these cells. This effect is not only greater in cells carrying a KRAS mutation compared to wildtype cells, but the KRAS mutant cells also have increased cell viability compared to wildtype cells. We hypothesize that the difference in cell viability and autophagy expression between KRAS mutant and KRAS wildtype cells after treatment with erlotinib can be of therapeutic value to treat CRC patients carrying KRAS mutations.

Inhibition of DUSP18 impairs cholesterol biosynthesis and promotes anti-tumor immunity in colorectal cancer

Tumor cells reprogram their metabolism to produce specialized metabolites that both fuel their own growth and license tumor immune evasion. However, the relationships between these functions remain poorly understood. Here, we report CRISPR screens in a mouse model of colo-rectal cancer (CRC) that implicates the dual specificity phosphatase 18 (DUSP18) in the establishment of tumor-directed immune evasion. Dusp18 inhibition reduces CRC growth rates, which correlate with high levels of CD8+ T cell activation. Mechanistically, DUSP18 dephosphorylates and stabilizes the USF1 bHLH-ZIP transcription factor. In turn, USF1 induces the SREBF2 gene, which allows cells to accumulate the cholesterol biosynthesis intermediate lanosterol and release it into the tumor microenvironment (TME). There, lanosterol uptake by CD8+ T cells suppresses the mevalonate pathway and reduces KRAS protein prenylation and function, which in turn inhibits their activation and establishes a molecular basis for tumor cell immune escape. Finally, the combination of an anti-PD-1 antibody and Lumacaftor, an FDA-approved small molecule inhibitor of DUSP18, inhibits CRC growth in mice and synergistically enhances anti-tumor immunity. Collectively, our findings support the idea that a combination of immune checkpoint and metabolic blockade represents a rationally-designed, mechanistically-based and potential therapy for CRC.

Exploring KRas Protein Dynamics: An Integrated Molecular Dynamics Analysis of KRas Wild and Mutant Variants.

This study employs a comprehensive approach combining protein retrieval, sequence alignment, and molecular dynamics simulations to investigate the structural dynamics and stability of wild-type KRas and its mutated variants (G12C, G12D, G12V, and G13D). The selected protein structures were retrieved from the Protein Data Bank (PDB) and prepared by using visual molecular dynamics (VMD) software. Sequence alignment using Clustal Omega provided a detailed comparison of the amino acid sequences, focusing on key mutation sites. Molecular dynamics simulations, performed with Gromacs, revealed distinct conformational changes and stability patterns in the wild-type and mutated KRas proteins over 100 ns. Clustering analysis identified higher conformational changes in the second α-helix of the mutated variants. The root-mean-square deviation (RMSD) distribution analysis showed variant-specific conformational dynamics, with G12V and G12D exhibiting slightly higher average RMSD values. Furthermore, clustering and RMSD analyses of specific amino acid residues (12, 13, 51, and 118) highlighted their roles in maintaining overall stability and influencing structural dynamics. The results indicate that mutations at positions 12 and 13 disrupt normal cycling between wild and mutated variants, leading to the persistent activation of KRas. Additionally, principal component analysis (PCA) elucidated unique conformational dynamics in mutated variants. Free energy landscape (FEL) analysis revealed alterations in the thermodynamic stability of mutated variants compared with the wild type. Overall, this study provides a detailed understanding of the structural changes associated with oncogenic mutations in KRas, offering insights crucial for targeted therapeutic strategies in KRas-driven cancers.

Tunneling nanotubes mediate KRas transport: Inducing tumor heterogeneity and altering cellular membrane mechanical properties

Mutation in oncogene KRas plays a crucial role in the occurrence and progression of numerous malignant tumors. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. We hypothesize that oncogene KRas mutations are intrinsic to alterations in cellular mechanics that promote malignant tumor generation and progression. Here, we demonstrate the use of optical tweezers coupled with a confocal fluorescence imaging system and gene interference technique to reveal that the mutant KRas protein can be transported between homogeneous and heterogeneous tumor cells by tunneling nanotubes (TNTs), resulting in a significant reduction of membrane tension and acceleration of membrane phospholipid flow in the recipient cells. Simultaneously, the changes in membrane mechanical properties of the tumor cells also enhance the metastatic and invasive ability of the tumors, which further contribute to the deterioration of the tumors. This finding helps to clarify the association between oncogene mutations and changes in the mechanical properties of tumor cells, which provides a theoretical basis for the development of cancer treatment strategies. Statement of significanceHere, we present a laser confocal fluorescence system integrated with optical tweezers to observe the transfer of mutant KRasG12D protein from mutant cells to wild-type cells through TNTs. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. Our results demonstrate a significant decrease in membrane tension and an increase in membrane phospholipid flow in recipient cells. These alterations in mechanical properties augment the migration and invasive capabilities of tumor cells, contributing to tumor malignancy. Our findings propose that cellular mechanical properties could serve as new markers for tumor development, and targeting membrane tension may hold potential as a therapeutic strategy.

Grifolin Induces Cell Death of Human Lung Cancer A549 Cell Line via Inhibiting KRAS-Mediated Multiple Signaling Pathways.

In the current work, grifolin was obtained from the twigs and leaves of Daphne genkwa for the first time and displayed significant growth inhibition against human lung carcinoma A549 cells. Subsequent in vitro antitumor evaluation revealed that grifolin could induce remarkable cell apoptosis and G0/G1 phase arrest, as well as block cell migration and invasion. In addition, grifolin also disrupted cellular energy metabolism by inducing reactive oxygen species, reducing adenosine triphosphate and mitochondrial membrane potential, and damaging DNA synthesis. Further RNA-seq analysis demonstrated that treatment of grifolin on A549 cells led to gene enrichment in MAPK, PI3K/Akt and NF-κB signaling pathways, all of which were inhibited by grifolin according to immunoblotting experiments. Further mechanistical studies disclosed that the expression of a key upstream protein KRAS was also blocked, and the cell death triggered by grifolin could be rescued by a RAS activator ML-099. Moreover, pretreatment of ML-099 on A549 cells could reverse the grifolin-induced downregulation of key proteins in the three aforementioned pathways. These findings indicate that grifolin could induce cell death in A549 cell line by inhibiting KRAS-mediated multiple signaling pathways.

Assessment of KRASG12C inhibitors for colorectal cancer.

Colorectal cancer (CRC) is a highly prevalent and lethal cancer worldwide. Approximately 45% of CRC patients harbor a gain-in-function mutation in KRAS. KRAS is the most frequently mutated oncogene accounting for approximately 25% of all human cancers. Gene mutations in KRAS cause constitutive activation of the KRAS protein and MAPK/AKT signaling, resulting in unregulated proliferation and survival of cancer cells and other aspects of malignant transformation, progression, and metastasis. While KRAS has long been considered undruggable, the FDA recently approved two direct acting KRAS inhibitors, Sotorasib and Adagrasib, that covalently bind and inactivate KRASG12C. Both drugs showed efficacy for patients with non-small cell lung cancer (NSCLC) diagnosed with a KRASG12C mutation, but for reasons not well understood, were considerably less efficacious for CRC patients diagnosed with the same mutation. Thus, it is imperative to understand the basis for resistance to KRASG12C inhibitors, which will likely be the same limitations for other mutant specific KRAS inhibitors in development. This review provides an update on clinical trials involving CRC patients treated with KRASG12C inhibitors as a monotherapy or combined with other drugs. Mechanisms that contribute to resistance to KRASG12C inhibitors and the development of novel RAS inhibitors with potential to escape such mechanisms of resistance are also discussed.

Identification of Matrine as a Kirsten rats Arcomaviral oncogene homolog inhibitor alleviating chemotherapy-induced neuropathic pain

BackgroundChemotherapy-induced peripheral neuropathy (CIPN) represents a prevailing and severe clinical concern, characterized by limited availability of clinically effective treatment strategies. Current evidence endorses matrine's potential as a neuroprotective and analgesic agent for CIPN. Nevertheless, the precise targets and mechanisms of action of matrine remain insufficiently explored, impeding comprehensive pharmacological investigation and clinical application. ObjectiveThis study endeavors to elucidate the analgesic and neuroprotective effects of matrine in mice with vincristine-induced neuropathic pain. A focal point is the identification of matrine's specific target and the underlying molecular mechanisms governing its analgesic and neuroprotective actions. MethodsTo discern matrine's analgesic effects in CIPN mice, we conducted behavioral experiments encompassing the Von Frey filament test and Hargreaves Test. Furthermore, we conducted electrophysiological and histopathological assessments involving HE staining, Nissl staining, and Fluoro-Jade B staining to evaluate matrine's effects on neuroprotection within dorsal root ganglia and the spinal cord of CIPN mice. Sequentially, thermal shift assay, GTP hydrolysis assay, and nucleotide exchange assay were executed to validate matrine's inhibitory effects on KRAS. Molecular docking and site-directed mutagenesis experiments were implemented to identify the precise binding pocket of matrine on KRAS. Lastly, matrine's inhibitory effects on downstream signaling pathways of KRAS were confirmed through experiments conducted at animal model. ResultsMatrine exhibited a notable increase in mechanical withdrawal threshold and thermal withdrawal latency in vincristine-treated mice. This compound substantially ameliorated the neurofunctional blockade associated with sensory and motor functions induced by vincristine. Moreover, matrine mitigated pathological damage within DRG and the L4-L5 spinal cord regions. The study's MST experiments indicated matrine's substantial elevation of KRAS's melting temperature. The GTP hydrolysis and nucleotide exchange assays revealed concentration-dependent inhibition of KRAS activity by matrine. Molecular docking provided insight into the binding mode of matrine with KRAS, while site-directed mutagenesis verified the specific binding site of matrine on KRAS. Lastly, matrine's inhibition of downstream Raf/Erk1/2 and PI3K/Akt/mTOR signaling pathways of KRAS was confirmed in VCR mice. ConclusionCompared to previous studies, our research has identified matrine as a natural inhibitor of the elusive protein KRAS, often considered "undruggable." Furthermore, this study has revealed that matrine exerts its therapeutic effects on chemotherapy-induced peripheral neuropathy (CIPN) by inhibiting KRAS activation, subsequently suppressing downstream signaling pathways such as Raf/Erk1/2 and PI3K/Akt/mTOR. This investigation signifies the discovery of a novel target for matrine, thus expanding the potential scope of its involvement in KRAS-related biological functions and diseases. These findings hold the promise of providing a crucial experimental foundation for forthcoming drug development initiatives centered around matrine, thereby advancing the field of pharmaceutical research.

Bioactive Compounds in Citrus reticulata Peel Are Potential Candidates for Alleviating Physical Fatigue through a Triad Approach of Network Pharmacology, Molecular Docking, and Molecular Dynamics Modeling

Physical fatigue (peripheral fatigue), which affects a considerable portion of the world population, is a decline in the ability of muscle fibers to contract effectively due to alterations in the regulatory processes of muscle action potentials. However, it lacks an efficacious therapeutic intervention. The present study explored bioactive compounds and the mechanism of action of Citrus reticulata peel (CR-P) in treating physical fatigue by utilizing network pharmacology (NP), molecular docking, and simulation-based molecular dynamics (MD). The bioactive ingredients of CR-P and prospective targets of CR-P and physical fatigue were obtained from various databases. A PPI network was generated by the STRING database, while the key overlapping targets were analyzed for enrichment by adopting KEGG and GO. The binding affinities of bioactive ingredients to the hub targets were determined by molecular docking. The results were further validated by MD simulation. Five bioactive compounds were screened, and 56 key overlapping targets were identified for CR-P and physical fatigue, whereas the hub targets with a greater degree in the PPI network were AKT1, TP53, STAT3, MTOR, KRAS, HRAS, JAK2, IL6, EGFR, and ESR1. The findings of the enrichment analysis indicated significant enrichment of the targets in three key signaling pathways, namely PI3K-AKT, MAPK, and JAK-STAT. The molecular docking and MD simulation results revealed that the bioactive compounds of CR-P exhibit a stronger affinity for interacting with the hub targets. The present work suggests that bioactive compounds of CR-P, specifically Hesperetin and Sitosterol, may ameliorate physical fatigue via the PI3K-AKT signaling pathway by targeting AKT1, KRAS, and MTOR proteins.

Identifying Potential SOS1 Inhibitors via Virtual Screening of Multiple Small Molecule Libraries against KRAS-SOS1 Interaction.

The RAS-MAPK signaling pathway, crucial for cell proliferation and differentiation, involves key proteins KRAS and SOS1. Mutations in the KRAS and SOS1 genes are implicated in various cancer types, including pancreatic, lung, and juvenile myelomonocytic leukemia. There is considerable interest in identifying inhibitors targeting KRAS and SOS1 to explore potential therapeutic strategies for cancer treatment. In this study, advanced in silico techniques were employed to screen small molecule libraries at this interface, leading to the identification of promising lead compounds as potential SOS1 inhibitors. Comparative analysis of the average binding free energies of these predicted potent compounds with known SOS1 small molecule inhibitors revealed that the identified compounds display similar or even superior predicted binding affinities compared to the known inhibitors. These findings offer valuable insights into the potential of these compounds as candidates for further development as effective anti-cancer agents.

Endogenous oncogenic KRAS expression increases cell proliferation and motility in near-diploid hTERT RPE-1 cells

About 18% of all human cancers carry a mutation in the KRAS gene making it among the most sought-after anti-cancer targets. However, mutant KRas protein has proved remarkably undruggable. The recent approval of the first generation of RAS inhibitors therefore marks a seminal milestone in the history of cancer research. Inevitably though, it also raises the predictable challenges of limited drug efficacies and acquired resistance. Hence, new approaches that improve our understanding of the tumorigenic mechanisms of oncogenic RAS within more physiological settings continue to be essential. Here, we have employed the near-diploid human hTERT RPE-1 cells to generate isogenic cell lines in which one of the endogenous KRAS alleles carries an oncogenic KRAS mutation at glycine 12. Cells with a KRASG12V/+, KRASG12C/+, or KRASG12D/+ genotype, together with wild-type KRASG12G(WT)/+ cells, reveal that oncogenic KRAS.G12X mutations increase cell proliferation rate and cell motility and reduced focal adhesions in KRASG12V/+ cells. EGF-induced phosphorylation of ERK and AKT was comparable between KRASG12V/+, KRASG12C/+, KRASG12D/+, and KRASG12G(WT)/+ cells. Interestingly, KRASG12X/+ cells showed varying responses to distinct inhibitors with the KRASG12V/+ and KRASG12D/+ cells more sensitive to hydroxyurea and MEK inhibitors, U0126 and trametinib, but more resistant to PI3K inhibitor, PIK-90, than the KRASG12G(WT)/+ cells. A combination of low doses of hydroxyurea and U0126 showed an additive inhibition on growth rate that was greater in KRASG12V/+ than wild-type cells. Collectively, these cell lines will be a valuable resource for studying oncogenic RAS signalling and developing effective anti-KRAS reagents with minimum cytotoxicity on wild-type cells.

The KRAS, ATR and CHEK1 expression levels in endometrial cancer are the risk factors predicting recurrence.

Endometrial cancer is the most prevalent gynecologic malignancy with a high risk of recurrence. Local recurrence occurs in 7-20% of patients with treated stage I cancer within 3 years after primary treatment. In this study, we found significantly elevated mRNA expression levels of the oncoprotein KRAS, along with two replicative stress markers, ATR and CHEK1, in samples of endometrial carcinomas of endometrium (ECE) from patients with relapse. In contrast, mRNA expression levels of the studied genes were low and uniform in samples from patients without relapse. Elevated levels of KRAS protein and the phosphorylated form of ATR/CHEK1 were distinguishing features of recurrent ECE. A strong positive correlation was found between elevated mRNA and protein levels of the studied molecules. Elevated KRAS protein levels are characteristic of poorly differentiated (G3) endometrial carcinomas with deep myometrial invasion in patients without recurrence. In contrast, in patients with recurrence, higher protein levels of KRAS, pATR and pCHEK1 were observed in samples of G1-2 endometrial carcinomas, with statistically significant differences confirmed for pATR. High pCHEK1 protein levels are associated with deep tumor invasion in the myometrium among patients with recurrence. ROC analysis confirmed that evaluating the specificity and sensitivity of KRAS, pATR and pCHEK1 predicts recurrence development in patients with ECE. Our findings indicate that markers of replicative stress may play a significant role in ECE pathogenesis. Determining their levels in tumor samples after primary treatment could help define patients at high risk of recurrence and guide consequent courses of treatment.

Targeting the molecular chaperone CCT2 inhibits GBM progression by influencing KRAS stability

Proper protein folding relies on the assistance of molecular chaperones post-translation. Dysfunctions in chaperones can cause diseases associated with protein misfolding, including cancer. While previous studies have identified CCT2 as a chaperone subunit and an autophagy receptor, its specific involvement in glioblastoma remains unknown. Here, we identified CCT2 promote glioblastoma progression. Using approaches of coimmunoprecipitation, mass spectrometry and surface plasmon resonance, we found CCT2 directly bound to KRAS leading to increased stability and upregulated downstream signaling of KRAS. Interestingly, we found that dihydroartemisinin, a derivative of artemisinin, exhibited therapeutic effects in a glioblastoma animal model. We further demonstrated direct binding between dihydroartemisinin and CCT2. Treatment with dihydroartemisinin resulted in decreased KRAS expression and downstream signaling. Highlighting the significance of CCT2, CCT2 overexpression rescued the inhibitory effect of dihydroartemisinin on glioblastoma. In conclusion, the study demonstrates that CCT2 promotes glioblastoma progression by directly binding to and enhancing the stability of the KRAS protein. Additionally, dihydroartemisinin inhibits glioblastoma by targeting the CCT2 and the following KRAS signaling. Our findings overcome the challenge posed by the undruggable nature of KRAS and offer potential therapeutic strategies for glioblastoma treatment.

Protein Ensemble Generation Through Variational Autoencoder Latent Space Sampling.

Mapping the ensemble of protein conformations that contribute to function and can be targeted by small molecule drugs remains an outstanding challenge. Here, we explore the use of variational autoencoders for reducing the challenge of dimensionality in the protein structure ensemble generation problem. We convert high-dimensional protein structural data into a continuous, low-dimensional representation, carry out a search in this space guided by a structure quality metric, and then use RoseTTAFold guided by the sampled structural information to generate 3D structures. We use this approach to generate ensembles for the cancer relevant protein K-Ras, train the VAE on a subset of the available K-Ras crystal structures and MD simulation snapshots, and assess the extent of sampling close to crystal structures withheld from training. We find that our latent space sampling procedure rapidly generates ensembles with high structural quality and is able to sample within 1 Å of held-out crystal structures, with a consistency higher than that of MD simulation or AlphaFold2 prediction. The sampled structures sufficiently recapitulate the cryptic pockets in the held-out K-Ras structures to allow for small molecule docking.