Ras Complex Research Articles

Allosteric nanobodies to study the interactions between SOS1 and RAS

Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse nanobodies that either stabilize or disrupt PPIs in a single experiment. We apply ChILL and DisCO to identify competitive, connective, or fully allosteric nanobodies that inhibit or facilitate the formation of the SOS1•RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro as well as RAS signalling in cellulo. One of these connective nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as pharmacophore for extending the repertoire of potential leads.

A Method to Conditionally Measure Target Engagement at Intracellular RAS and RAF Complexes.

Dysfunction of the RAS/mitogen-activated protein kinase (MAPK) pathway is a common driver of human cancers. As such, both the master regulator of the pathway, RAS, and its proximal kinase effectors, RAFs, have been of interest as drug targets for decades. Importantly, signaling within the RAS/MAPK pathway is highly coordinated due to the formation of a higher-order complex called the RAS/RAF signalosome, which may minimally contain dimers of both RAS and RAF protomers. In the disease state, RAS and RAF assemble in homo- and/or heterodimeric forms. Traditionally, drug development campaigns for both RAS and RAF have utilized biochemical assays of purified recombinant protein. As these assays do not query the RAS or RAF proteins in their full-length and complexed forms in cells, potency results collected using these assays have often failed to correlate with inhibition of the MAPK pathway. To more accurately quantify engagement at this signaling components, we present a bioluminescence resonance energy transfer (BRET)-based method to conditionally measure target engagement at individual protomers within the RAS/RAF signalosome in live cells.

Abstract 2619: Characterization of RAS assembly architectures in living cells

Abstract Assembly of RAS proteins and RAS effectors into “nanoclusters” at the cell membrane regulates RAS signaling output. The structure and composition of membrane-bound RAS complexes is dynamic, but defined states appear to exist. We previously found biological and biophysical evidence for two different models of membrane-bound RAS assemblies where RAS proteins form symmetric or asymmetric RAS-RAS interactions. It is unknown what functions these two architectures might serve and what determinants these nanoclusters might be subject to. To further characterize which architectural states occur in various biological scenarios, we developed a plate-based assay to detect RAS-RAS interactions in cells. The system controls for RAS expression and can be used to discriminate symmetric and asymmetric RAS-RAS interactions. It can also be used to assess the relative contributions of the G domain (1-166) and hypervariable region (HVR) in mediating RAS-RAS interactions. We found that RAS isoforms can homo- and hetero-dimerize and observed that the G domain contributes to RAS-RAS interactions, but that the HVR does not. We also found that the primary determinant of HVR interaction with the membrane is electrostatic charge. Regarding cancer-causing mutations, we found that in the KRAS G12D context, RAS-RAS interactions appear to favor asymmetric RAS-RAS interactions while KRAS Q61H favors symmetric RAS-RAS interactions. Citation Format: Zhiwei Zhou, Xing-Xiao Li, Cynthia Meyer, Yu-An Zhang, Deekshi Angira, Kenneth Westover. Characterization of RAS assembly architectures in living cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2619.

Predicting membrane orientational states of RAS and RAS-RAF based on the lipid environment using deep learning.

Cells utilize different active mechanisms to regulate cellular growth, differentiation, and survival. RAS and RAF proteins play a central role in these mechanisms, which are regulated by complex protein-lipid interactions. It is critical to identify molecular details by which local lipid environment modulates the behavior of these proteins to have detailed understanding of the mechanism underlying cancer formation. Here, we first employed deep learning (DL) to identify orientational states of RAS proteins and associated lipids using an extensive training data from a massive simulation campaign of 120,000 coarse-grained MD simulations each over 1 microsecond long, run using our MuMMI. Our results show that our DL model can predict protein states with an accuracy of over 80% based on the underlying lipid fingerprints and all the different lipid species contribute to the preferred RAS membrane orientational states.For the more complex RAS-RAF proteins, we employed DL (ResNet model) to explore the relationship between protein conformational structures and local lipids by predicting orientational states of RAS and RAS-RAF protein complexes based on the lipid densities around the protein domains. We obtained our training data from a recent RAS-RAF MuMMI campaign containing over 96 ms of aggregated CG simulation time. Our findings demonstrate that there is a significant correlation between the RAS and RAS-RAF orientational states and specific lipid densities around the protein domains. Our ResNet model can predict six protein states with an overall accuracy of around 80% from lipid density data. The findings of this work can assist in designing novel therapies targeting critical protein-lipid interactions involved in the mechanisms associated with cancer development. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC5207NA27344. Release:LLNL-ABS-840775.

Afadin couples RAS GTPases to the polarity rheostat Scribble

AFDN/Afadin is required for establishment and maintenance of cell-cell contacts and is a unique effector of RAS GTPases. The biological consequences of RAS complex with AFDN are unknown. We used proximity-based proteomics to generate an interaction map for two isoforms of AFDN, identifying the polarity protein SCRIB/Scribble as the top hit. We reveal that the first PDZ domain of SCRIB and the AFDN FHA domain mediate a direct but non-canonical interaction between these important adhesion and polarity proteins. Further, the dual RA domains of AFDN have broad specificity for RAS and RAP GTPases, and KRAS co-localizes with AFDN and promotes AFDN-SCRIB complex formation. Knockout of AFDN or SCRIB in epithelial cells disrupts MAPK and PI3K activation kinetics and inhibits motility in a growth factor-dependent manner. These data have important implications for understanding why cells with activated RAS have reduced cell contacts and polarity defects and implicate AFDN as a genuine RAS effector.

Structural basis for SHOC2 modulation of RAS signalling

The RAS–RAF pathway is one of the most commonly dysregulated in human cancers1–3. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer5 and active RAF dimer5–8 bound to 14-3-39,10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS11–13, but the structure of SHOC2 and the architecture of the presumptive SHOC2–PP1C–RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2–PP1C–MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

The glucocorticoid receptor associates with RAS complexes to inhibit cell proliferation and tumor growth.

Mutations that activate members of the RAS family of GTPases are associated with various cancers and drive tumor growth. The glucocorticoid receptor (GR), a member of the nuclear receptor family, has been proposed to interact with and inhibit the activation of components of the PI3K-AKT and MAPK pathways downstream of RAS. In the absence of activating ligands, we found that GR was present in cytoplasmic KRAS-containing complexes and inhibited the activation of wild-type and oncogenic KRAS in mouse embryonic fibroblasts and human lung cancer A549 cells. The DNA binding domain of GR was involved in the interaction with KRAS, but GR-dependent inhibition of RAS activation did not depend on the nuclear translocation of GR. The addition of ligand released GR-dependent inhibition of RAS, AKT, the MAPK p38, and the MAPKK MEK. CRISPR-Cas9-mediated deletion of GR in A549 cells enhanced tumor growth in xenografts in mice. Patient samples of non-small cell lung carcinomas showed lower expression of NR3C1, the gene encoding GR, compared to adjacent normal tissues and lower NR3C1 expression correlated with a worse disease outcome. These results suggest that glucocorticoids prevent the ability of GR to limit tumor growth by inhibiting RAS activation, which has potential implications for the use of glucocorticoids in patients with cancer.

ANALYSIS OF THE SEISMIC AND IONOSPHERIC EFFECTS OF THE KUDARINSKY EARTHQUAKE ON DECEMBER 9, 2020

According to the data obtained on the equipment of the IEC SB RAS complex monitoring base for hazardous geological processes "Buguldeika" (Shared Research Facilities "Geodynamics and Geochronology" of IEC SB RAS) and Shared Research Facilities "Angara" of ISTP SB RAS an analysis of the characteristics of the Kudarinsky earthquake (09.12.2020) and the behaviour of the ionosphere during this event was carried out. The source parameters of the earthquake were obtained – the seismic moment of the earthquake (M0=3.02·1017 N·m), the moment magnitude (Mw=5.6), the source radius (2.43 km), and the stress drop (1.26 MPa).The analysis of the ionosphere behaviour carried out using GPS/GLONASS receivers did not reveal disturbances caused by the Kudarinsky earthquake, which is most likely due to the relatively small magnitude of this earthquake. An analysis of the observation series related to the Kudarinsky earthquake showed the efficiency of using the Core Facilities Centre equipment and complex monitoring bases for studying seismicity, which is the most dangerous natural process for the Baikal region.

Clinical and Genetic Relevance of Somatic Mutations in Core Binding Factor (CBF) Acute Myeloid Leukemia (AML) Patients Using Serial Sequencing

ObjectivesTo investigate the clinical relevance of somatic mutations in core binding factor (CBF) acute myeloid leukemia (AML) patients at diagnosis and relapse and hierarchy of mutation acquisition using serial sequencing.Patients and MethodsEighty seven patients (pts) diagnosed with CBF AML were enrolled in this study (62 pts carrying RUNX1-RUNX1T1 /t(8; 21) and 25 pts carrying CBFB-MYH11 /inv(16). Median follow-up duration among survivors was 56.9 months (range 0-155.3). Using a 84 gene panel, we performed targeted deep sequencing in 357 samples using Illumina Hiseq 2500. Sequenced samples include bone-marrow/peripheral blood taken at initial diagnosis (n=87) and T-cell (CD3+, n=68), CD34+/CD38-(n=66) and CD34+/CD38+ (n=68) fractions. In addition, samples taken at complete remission (n=53), and relapse (n=15) were sequenced. Average of on-target coverage was 1657.8x.ResultsWe detected 166 mutations in 79/87 pts at time of diagnosis (90.8%, median 2 mutations/pts, range 0-7). At diagnosis, KIT (39%), NRAS (33%), ASXL2 (14%), KRAS (13%), RAD21 (7%), and FLT3 (6%) were commonly mutated. When grouped by biological pathway, frequencies of mutations in RAS (KRAS or NRAS ), chromatin modifiers and cohesin complex were significantly different among two sub-populations (p = 0.002, 0.01, and 0.02, respectively, Figure A). Survival analyses show that KIT -D816mut is an adverse prognostic factor for overall survival (hazard ratio (HR) 2.31, 95% CI [1.04-5.14], p=0.04) and relapse (HR 2.76 [1.06-7.16], p =0.04). Mutation in RAS was a favorable factor for relapse (HR 0.13 [0.03-0.56], p=0.001, Figure B). Multivariate analysis confirmed that only RAS mutation was a significant favorable factor for the incidence of relapse (HR 0.20 [0.05-0.86], p=0.03).Taking advantage of serial sequencing and real-time polymerase chain reaction (PCR) data (RUNX1-RUNX1T1 ), we inferred mutation dynamics and clonal hierarchies. Mean allelic burden at diagnosis and relapse were comparable (mean variant allele frequency (VAF) = 22.17% and 22.22%, respectively), whereas mutations were nearly cleared at complete remission (mean VAF = 0.25%) (Figure C). With initial genetic rearrangement detected at both diagnosis and relapse, 14/15 pts carried additional 39 mutations throughout the course of the disease. Fifteen mutations were stable, whereas 9 and 15 mutations were cleared/decreased and acquired/selected at relapse. Inference based on mutation dynamics revealed stable mutations are more likely to be earlier events than mutations that are cleared at relapse (p = 0.01, Figure D). Furthermore, integrated analyses of reduction rate of the RUNX1-RUNX1T1 and mutation dynamics showed RUNX1-RUNX1T1 is no later event than all targeted mutations in our cohort, further explaining clonal hierarchies of CBF AML with RUNX1-RUNX1T1(Figure E).Conclusion and SummaryCurrent study provided distinct mutation patterns between two sub-population of CBF AML. Multivariate analyses showed that presence of mutation in KRAS or NRAS was a favorable prognostic factor. In addition, results from longitudinal sequencing combined with RUNX1-RUNX1T1 level provides further insights on the order of genetic mutation acquisition during leukemogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Venetoclax with azacitidine or decitabine in blast-phase myeloproliferative neoplasm: A multicenter series of 32 consecutive cases.

Venetoclax (Ven) combined with a hypomethylating agent (HMA) has now emerged as an effective treatment regimen for acute myeloid leukemia, in both de novo and relapsed/refractory setting. The current multicenter study retrospectively examined Ven + HMA treatment outcome among 32 patients (median age 69 years; 59% males) with blast‐phase myeloproliferative neoplasm (MPN‐BP). Pre‐leukemic phenotype included essential thrombocythemia (ET)/post‐ET myelofibrosis (34%), polycythemia vera (PV)/post‐PV myelofibrosis (38%) and primary myelofibrosis (28%). Twenty‐nine study patients were fully annotated cytogenetically and molecularly (NGS): 69% harbored complex karyotype and/or mutations, including TP53 (41%), IDH1/2 (21%), ASXL1 (21%), N/KRAS (14%), SRSF2 (10%), EZH2 (10%) and U2AF1 (7%). All patients received Ven combined with either azacitidine (n = 12) or decitabine (n = 20); either up front (n = 23) or after failing another induction therapy (n = 9). Complete remission with (CR) or without (CRi) count recovery was achieved in 14 (44%) patients and was more likely to occur in the absence of pre‐leukemic PV/post‐PV myelofibrosis phenotype (p < .01), complex karyotype (p < .01) or K/NRAS (p = .03) mutations; seven of eight patients (88%) without vs four of 21 (19%) with complex karyotype or K/NRAS mutation achieved CR/CRi (p < .01); all 11 informative patients with pre‐leukemic PV/post‐PV myelofibrosis phenotype displayed complex karyotype (p < .01). In contrast, neither TP53 (p = .45) nor IDH1/2 (p = .63) mutations affected response. Compared to historical controls treated with HMA alone (n = 26), the CR/CRi rate (44% vs 4%) and median survival (8 vs 5.5 months) were more favorable with Ven + HMA, but without significant difference in overall survival. Importantly, six patients with CR/CRi subsequently received allogeneic hematopoietic stem cell transplant (AHSCT). Note, Ven + HMA produces robust CR/CRi rates in MPN‐BP, especially in the absence of RAS mutations and complex karyotype, thus enabling AHSCT, in some patients.

RAS Nanoclusters: Dynamic Signaling Platforms Amenable to Therapeutic Intervention.

RAS proteins are mutated in approximately 20% of all cancers and are generally associated with poor clinical outcomes. RAS proteins are localized to the plasma membrane and function as molecular switches, turned on by partners that receive extracellular mitogenic signals. In the on-state, they activate intracellular signal transduction cascades. Membrane-bound RAS molecules segregate into multimers, known as nanoclusters. These nanoclusters, held together through weak protein–protein and protein–lipid associations, are highly dynamic and respond to cellular input signals and fluctuations in the local lipid environment. Disruption of RAS nanoclusters results in downregulation of RAS-mediated mitogenic signaling. In this review, we discuss the propensity of RAS proteins to display clustering behavior and the interfaces that are associated with these assemblies. Strategies to therapeutically disrupt nanocluster formation or the stabilization of signaling incompetent RAS complexes are discussed.

Ras–guanine nucleotide complexes: A UV spectral deconvolution method to analyze protein concentration, nucleotide stoichiometry, and purity

The many members of the Ras superfamily are small GTPases that serve as molecular switches. These proteins bind the guanine nucleotides GTP and GDP with picomolar affinities, thereby stabilizing on- and off-signaling states, respectively. Quantitative in vitro Ras studies require accurate determination of total protein, its fractional occupancy with guanine nucleotide, and spectroscopic purity. Yet the high nucleotide affinity of Ras and the overlapping UV spectra of the protein and bound nucleotide make such determinations challenging. Here we describe a generalizable UV spectral deconvolution method to analyze the total protein concentration, total nucleotide stoichiometry, and purity of Ras complexes.

Analyzing solar, cosmic, and geophysical events in September 2017 using SHISRA SB RAS complex observations

We report the results of monitoring of cosmic rays and geomagnetic field along 210 magnetic meridians in Yakutia in the first half of September 2017. The energy spectrum of solar cosmic rays during Ground Level Enhancement in September 10, 2017 is estimated as J=3027E–1.99exp(–E/729 MeV). We present the results of the forecast and complex analysis of the magnetic storm on September 7–9, 2017 with Dst=–124 nT. The forecast lead time is about one day. We examine how the storm affected the electric potential and VLF signal propagation from RSDN-20 radio navigation stations. Irregular Pi3–Pi1 pulsations occurred during the September 8, 2017 magnetic storm from 12 to 20 UT. The pulsations were accompanied by variations in electrotelluric potentials and geomagnetic fields with the correlation coefficient between them ρ(E, H)=0.5÷0.9. The effects of the magnetic storm manifested themselves as an increase in the attenuation and a decrease in the phase delay of VLF radio signals.

High-throughput screening identifies small molecules that bind to the RAS:SOS:RAS complex and perturb RAS signaling

K-RAS is mutated in approximately 30% of human cancers, resulting in increased RAS signaling and tumor growth. Thus, RAS is a highly validated therapeutic target, especially in tumors of the pancreas, lung and colon. Although directly targeting RAS has proven to be challenging, it may be possible to target other proteins involved in RAS signaling, such as the guanine nucleotide exchange factor Son of Sevenless (SOS). We have previously reported on the discovery of small molecules that bind to SOS1, activate SOS-mediated nucleotide exchange on RAS, and paradoxically inhibit ERK phosphorylation (Burns et al., PNAS, 2014). Here, we describe the discovery of additional, structurally diverse small molecules that also bind to SOS1 in the same pocket and elicit similar biological effects. We tested >160,000 compounds in a fluorescence-based assay to assess their effects on SOS-mediated nucleotide exchange. X-Ray structures revealed that these small molecules bind to the CDC25 domain of SOS1. Compounds that elicited high levels of nucleotide exchange activity in vitro increased RAS-GTP levels in cells, and inhibited phospho ERK levels at higher treatment concentrations. The identification of structurally diverse SOS1 binding ligands may assist in the discovery of new molecules designed to target RAS-driven tumors.

THE LONGITUDINAL STUDY RESULTS OF THE PHYSIOLOGICAL STATUS OF MALE NORTHERNERS - PARTICIPANTS OF THE PROJECT “MARS-500”

We studied physiological status and somatic health of 23 male volunteers (age 24-50 yrs) recruited as a "northern" control group for the experiment "Mars-500". The study was performed using standardized methods, established by the Institute of Medical and Biological Problems of the RAS, using hardware and software complex "Ecosan-2007". Repetitive experiments were carried out in 6 years. Collected data were processed using Biostat and Statistica statistical software. In the course of the study we found certain features in the physiological status and the somatic health of the participants related to their inhabitation in the North. We discovered that our volunteers had decreased somatic health levels according to the Apanasenko scale. Analyzing the heart rate variability in our subjects we observed tension in regulatory systems and a domination of the central regulation circuit over the autonomous circuit. Additionally our subjects showed a deterioration of the hemodynamics regulation under a physical load. After 6 years we observed statistically significant increases in the body mass, the heart rate after physical load, biological age, and significant decrease of the physical health, which indicate the accelerated ageing in the group of surveyed. A number of persons with premorbid status have increased, indicating the increased tension of the regulatory systems and the decrease in the adaptability potential.

Modeling of RAS complexes supports roles in cancer for less studied partners

BackgroundRAS protein interactions have predominantly been studied in the context of the RAF and PI3kinase oncogenic pathways. Structural modeling and X-ray crystallography have demonstrated that RAS isoforms bind to canonical downstream effector proteins in these pathways using the highly conserved switch I and II regions. Other non-canonical RAS protein interactions have been experimentally identified, however it is not clear whether these proteins also interact with RAS via the switch regions.ResultsTo address this question we constructed a RAS isoform-specific protein-protein interaction network and predicted 3D complexes involving RAS isoforms and interaction partners to identify the most probable interaction interfaces. The resulting models correctly captured the binding interfaces for well-studied effectors, and additionally implicated residues in the allosteric and hyper-variable regions of RAS proteins as the predominant binding site for non-canonical effectors. Several partners binding to this new interface (SRC, LGALS1, RABGEF1, CALM and RARRES3) have been implicated as important regulators of oncogenic RAS signaling. We further used these models to investigate competitive binding and multi-protein complexes compatible with RAS surface occupancy and the putative effects of somatic mutations on RAS protein interactions.ConclusionsWe discuss our findings in the context of RAS localization to the plasma membrane versus within the cytoplasm and provide a list of RAS protein interactions with possible cancer-related consequences, which could help guide future therapeutic strategies to target RAS proteins.

Exploring the interactions of the RAS family in the human protein network and their potential implications in RAS-directed therapies.

RAS proteins are the founding members of the RAS superfamily of GTPases. They are involved in key signaling pathways regulating essential cellular functions such as cell growth and differentiation. As a result, their deregulation by inactivating mutations often results in aberrant cell proliferation and cancer. With the exception of the relatively well-known KRAS, HRAS and NRAS proteins, little is known about how the interactions of the other RAS human paralogs affect cancer evolution and response to treatment. In this study we performed a comprehensive analysis of the relationship between the phylogeny of RAS proteins and their location in the protein interaction network. This analysis was integrated with the structural analysis of conserved positions in available 3D structures of RAS complexes. Our results show that many RAS proteins with divergent sequences are found close together in the human interactome. We found specific conserved amino acid positions in this group that map to the binding sites of RAS with many of their signaling effectors, suggesting that these pairs could share interacting partners. These results underscore the potential relevance of cross-talking in the RAS signaling network, which should be taken into account when considering the inhibitory activity of drugs targeting specific RAS oncoproteins. This study broadens our understanding of the human RAS signaling network and stresses the importance of considering its potential cross-talk in future therapies.

Atorvastatin inhibits insulin synthesis by inhibiting the Ras/Raf/ERK/CREB pathway in INS-1 cells.

Backround:Type 2 diabetes has become a global epidemic disease. Atorvastatin has become a cornerstone in the prevention and treatment of atherosclerosis. However, increasing evidence showed that statins can dose-dependently increase the risk of diabetes mellitus. The mechanism is not clear.Objective:The Ras complex pathway (Ras/Raf/extracellular signal-regulated kinase [ERK]/cAMP response element-binding protein [CREB]) is the major pathway that regulates the gene transcription. Except for the inhibition of cholesterol synthesis by inhibiting the 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-COA) reductase, statins can also downregulate the phosphorylation of a series of downstream substrates including the key proteins of the Ras complex pathway, therefore may inhibit the insulin syntheses in pancreatic beta cells. In our study, we investigated the inhibitory effect and the underlying mechanism of atorvastatin on insulin synthesis in rat islets.Methods:Islets were isolated from Wistar rats and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. The insulin content in the medium was measured by radioimmunoassay before and after the treatment of 50 μM atorvastatin. Effect of atorvastatin on the expression of insulin message Ribonucleic acid (mRNA) in pancreatic islet beta cells was also detected using quantitative real-time polymerase chain reaction. Western blotting was used to explore the possible role of the Ras complex pathway (Ras/Raf/ERK/CREB) in atorvastatin-inhibited insulin synthesis. The effects of atorvastatin on the binding of nuclear transcription factor p-CREB with CRE in INS-1 cells were examined via chromatin immunoprecipitation assay.Results:Compared with the control group, the insulin level decreased by 27.1% at 24 hours after atorvastatin treatment. Atorvastatin inhibited insulin synthesis by decreasing insulin mRNA expression of pancreatic islet beta cells. The activities of Ras, Raf-1, and p-CREB in the Ras complex pathway were inhibited by 50 μM atorvastatin in INS-1 cells in vitro. Moreover, 50 μM atorvastatin reduced the binding of p-CREB with deoxyribonucleic acid (DNA) in INS-1 cells in vitro.Conclusion:Atorvastatin inhibits insulin synthesis in beta cells by inhibiting the activation of the Ras complex pathway.

Abstract IA13: Discovery of K-Ras inhibitors for the treatment of cancer

Abstract Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active receptor tyrosine kinases (RTKs) results in the deregulation of cellular signals governing growth and survival in cancer. The guanine nucleotide exchange factor Son of Sevenless (SOS) catalyzes the rate-limiting step in the activation of Ras by exchanging GDP for GTP. SOS is therefore a key control point for the propagation of RTK and Ras signaling. Here we report the discovery of small molecules that bind to a unique pocket on the Ras:SOS:Ras complex, increase SOScat-catalyzed nucleotide exchange, and perturb Ras signaling pathways in cells. X-ray crystallographic studies of Ras:SOS:Ras complexed with these small molecules reveal that they bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of the x-ray structures of multiple co-complexes. In addition, structure-based mutational analyses indicate that this newly identified pocket is essential for compound activity. As predicted, these molecules increase Ras-GTP levels in cells. However, they unexpectedly inhibit MAPK and PI3K signaling. Our studies suggest a novel way to target K-Ras and offer possible starting points for the discovery of compounds that could be used to treat Ras-driven tumors. Citation Format: Stephen W. Fesik. Discovery of K-Ras inhibitors for the treatment of cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA13. doi: 10.1158/1557-3125.RASONC14-IA13