Ras Regulation Research Articles

Regulation of RAS by ubiquitination

Signal Transduction The protein LZTR1 is mutated in human cancers and developmental diseases. Work from two groups now converges to implicate the protein in regulating signaling by the small guanosine triphosphatase RAS. Steklov et al. showed that mice haploinsufficient for LZTR1 recapitulated aspects of the human disease Noonan syndrome. Their biochemical studies showed that LZTR1 associated with RAS. LZTR1 appears to function as an adaptor that promotes ubiquitination of RAS, thus inhibiting its signaling functions. Bigenzahn et al. found LZTR1 in a screen for proteins whose absence led to resistance to the tyrosine kinase inhibitors used to treat cancers caused by the BCR-ABL oncogene product. Their biochemical studies and genetic studies in fruitflies also showed that loss of LZTR1 led to increased activity of RAS and signaling through the mitogen-activated protein kinase pathway. Science , this issue p. [1177][1], p. [1171][2] [1]: /lookup/doi/10.1126/science.aap7607 [2]: /lookup/doi/10.1126/science.aap8210

Hunting and fishing for cancer genes

Cancer Mucosal melanoma is a rare, but deadly, form of melanoma that occurs in sun-protected tissues. Little is known about the genetic alterations that drive the growth of these tumors. Ablain et al. sequenced mucosal melanomas from 43 patients and found that a substantial fraction showed inactivation or loss of SPRED1 , a gene that encodes a negative regulator of RAS–MAPK (mitogen-activated protein kinase) signaling. Using a platform called MAZERATI (Modeling Approach in Zebrafish for Rapid Tumor Initiation), they discovered that SPRED1 loss may help explain the poor response of melanoma patients to drugs that inhibit the KIT tyrosine kinase. The results suggest that a combination of KIT inhibitors and drugs that inhibit MAPK signaling may be more effective. Science , this issue p. [1055][1] [1]: /lookup/doi/10.1126/science.aau6509

LZTR1 is a regulator of RAS ubiquitination and signaling.

In genetic screens aimed at understanding drug resistance mechanisms in chronic myeloid leukemia cells, inactivation of the cullin 3 adapter protein-encoding leucine zipper-like transcription regulator 1 (LZTR1) gene led to enhanced mitogen-activated protein kinase (MAPK) pathway activity and reduced sensitivity to tyrosine kinase inhibitors. Knockdown of the Drosophila LZTR1 ortholog CG3711 resulted in a Ras-dependent gain-of-function phenotype. Endogenous human LZTR1 associates with the main RAS isoforms. Inactivation of LZTR1 led to decreased ubiquitination and enhanced plasma membrane localization of endogenous KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). We propose that LZTR1 acts as a conserved regulator of RAS ubiquitination and MAPK pathway activation. Because LZTR1 disease mutations failed to revert loss-of-function phenotypes, our findings provide a molecular rationale for LZTR1 involvement in a variety of inherited and acquired human disorders.

Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination.

The leucine zipper-like transcriptional regulator 1 (LZTR1) protein, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, is implicated in human disease, yet its mechanism of action remains unknown. We found that Lztr1 haploinsufficiency in mice recapitulates Noonan syndrome phenotypes, whereas LZTR1 loss in Schwann cells drives dedifferentiation and proliferation. By trapping LZTR1 complexes from intact mammalian cells, we identified the guanosine triphosphatase RAS as a substrate for the LZTR1-CUL3 complex. Ubiquitome analysis showed that loss of Lztr1 abrogated Ras ubiquitination at lysine-170. LZTR1-mediated ubiquitination inhibited RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations disrupted either LZTR1-CUL3 complex formation or its interaction with RAS proteins. RAS regulation by LZTR1-mediated ubiquitination provides an explanation for the role of LZTR1 in human disease.

Mutually inhibitory Ras-PI(3,4)P2 feedback loops mediate cell migration

Signal transduction and cytoskeleton networks in a wide variety of cells display excitability, but the mechanisms are poorly understood. Here, we show that during random migration and in response to chemoattractants, cells maintain complementary spatial and temporal distributions of Ras activity and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2]. In addition, depletion of PI(3,4)P2 by disruption of the 5-phosphatase, Dd5P4, or by recruitment of 4-phosphatase INPP4B to the plasma membrane, leads to elevated Ras activity, cell spreading, and altered migratory behavior. Furthermore, RasGAP2 and RapGAP3 bind to PI(3,4)P2, and the phenotypes of cells lacking these genes mimic those with low PI(3,4)P2 levels, providing a molecular mechanism. These findings suggest that Ras activity drives PI(3,4)P2 down, causing the PI(3,4)P2-binding GAPs to dissociate from the membrane, further activating Ras, completing a positive-feedback loop essential for excitability. Consistently, a computational model incorporating such a feedback loop in an excitable network model accurately simulates the dynamic distributions of active Ras and PI(3,4)P2 as well as cell migratory behavior. The mutually inhibitory Ras-PI(3,4)P2 mechanisms we uncovered here provide a framework for Ras regulation that may play a key role in many physiological processes.

Abstract PR12: A combined protein-protein interaction and genetic interaction map defines new and critical Kras effectors in non-small cell lung cancer

Abstract Despite intensive study, no drugs in clinical use specifically target KRAS-mutant tumors. Uncharacterized feedback mechanisms and parallel pathways have stymied the treatment of KRAS-mutant tumors with Raf and PI3K inhibitors, and the KRas protein itself does not easily accommodate binding of small-molecule inhibitors. These challenges demand more systematic and quantitative characterization of the physical and genetic relationships between Ras regulators and effectors. To that end, we used tandem affinity purification of Kras, Hras and Nras, their activated alleles and key proteins with known regulatory (GEFs, GAPs) or effector (Raf, RalGDS, RIN1/2) roles in both 293 cells and A549 NSCLC cells to generate a high-confidence protein-protein interaction (PPI) network. This map of 220 proteins and 1,400 physical interactions was used to design an sgRNA library with 10 guides/gene. This library was screened in Cas9-expressing A549 cells and grown for 14 days before analysis for dropout or enhanced representation of sgRNAs. Approximately 120 genes showed positive or negative growth effects. PPIs and genetic interactions (GIs) were cross-referenced with public PPI data and TCGA patient data to assemble a combined physical PPI and genetic map informed by cancer mutations. This map suggests many hypotheses for PPIs critical for growth control. This set was used to construct a sgRNA library covering 120 genes of probable relevance to the Ras pathway with ~60 “safe harbor” control sgRNAs. This library was screened in a two-cassette sgRNA system testing 14K pairwise genetic effects to identify quantitative changes in growth in A549 and H23 NSCLC lines. This screen showed >100 genetic interactions, which in conjunction with PPIs, identify coupling between the Raf/MEK/ERK kinase, Ral and Rap GTPase, RNA processing, and cell adhesion pathways. The screen identified new candidate effector pathways for cell adhesion, RNA processing, Rap GTPase regulation, and protein processing, including the RADIL, RGL, and RIN Kras effectors. Validation focused using the synthetic lethal interactions observed in the sgRNA screen to predict drug combinations showing drug synergy in A549 and H23 cells. Using 11-point dose titrations and isobologram analysis of drug combinations, we see strong synergy among PI3 kinase, Raf, and Erk inhibitors in these cells. Using the recently described Kras G12C inhibitor, expressed in H23 cells, we have validated that sgRNA deletion of the the key Kras effector for specific pathways including cell adhesion (RADIL), growth signaling (RAF), and endocytosis/ macropinocytosis (RIN) are affected and that use of the Kras inhibitor ARS-853 shows much reduced effects on specific Kras effector pathways in cells deleted for these effectors. These systematic data underscore the limitations of our current understanding of Kras-driven cancers, revealing new genetic vulnerabilities and target candidates. This abstract is also being presented as Poster A28. Citation Format: Marcus R. Kelly, Kyuho Han, Nancie Mooney, Edwin Jeng, Kaja Kostyrko, Alejandro Sweet-Cordero, Michael Bassik, Peter K. Jackson. A combined protein-protein interaction and genetic interaction map defines new and critical Kras effectors in non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR12.

Ras-Specific GTPase-Activating Proteins-Structures, Mechanisms, and Interactions.

Ras-specific GTPase-activating proteins (RasGAPs) down-regulate the biological activity of Ras proteins by accelerating their intrinsic rate of GTP hydrolysis, basically by a transition state stabilizing mechanism. Oncogenic Ras is commonly not sensitive to RasGAPs caused by interference of mutants with the electronic or steric requirements of the transition state, resulting in up-regulation of activated Ras in respective cells. RasGAPs are modular proteins containing a helical catalytic RasGAP module surrounded by smaller domains that are frequently involved in the subcellular localization or contributing to regulatory features of their host proteins. In this review, we summarize current knowledge about RasGAP structure, mechanism, regulation, and dual-substrate specificity and discuss in some detail neurofibromin, one of the most important negative Ras regulators in cellular growth control and neuronal function.

Abstract 1313: A combined protein-protein interaction and genetic interaction map defines new and critical Kras effectors in NSCLC

Abstract Despite intensive study, no drugs in clinical use specifically target KRAS-mutant tumors. Uncharacterized feedback mechanisms and parallel pathways have stymied the treatment of KRAS-mutant tumors with Raf and PI3K inhibitors, and the KRas protein itself does not easily accommodate binding of small-molecule inhibitors. These challenges demand more systematic and quantitative characterization of the physical and genetic relationships between Ras regulators and effectors. Thus we used tandem affinity purification of Kras, Hras and Nras, their activated alleles and key proteins with known regulatory (GEFs, GAPs) or effector (Raf, RalGDS) roles in both 293 cells and A549 NSCLC cells to generate a high-confidence protein-protein interaction (PPI) network. This map of 220 proteins and 1,400 physical interactions was used to design an sgRNA library with 10 guides/gene. This library was screened in Cas9-expressing A549 cells and grown for 14 days before analysis for dropout or enhanced representation of sgRNAs. Approximately 120 genes showed positive or negative growth effects. PPIs and genetic interactions (GIs) were cross-referenced with public PPI data and TCGA patient data to assemble a combined physical PPI and genetic map informed by cancer mutations. This map suggests many hypotheses for PPIs critical for growth control. This set was used to construct a sgRNA library covering 120 genes of probable relevance to the Ras pathway with ~60 “safe harbor” control sgRNAs. This library was screened in a two-cassette sgRNA system testing 14K pairwise genetic effects to identify quantitative changes in growth in A549 and H23 NSCLC lines. This screen showed >100 genetic interactions, which in conjunction with PPIs identify coupling between the Raf/MEK/ERK kinase, Ral and Rap GTPase, RNA processing and cell adhesion pathways. The screen identified new candidate effector pathways for cell adhesion, RNA processing, Rap GTPase regulation and protein processing, including the RADIL, RGL and RIN Kras effectors. Validation focused on using the synthetic lethal interactions observed in the sgRNA screen to predict drug combinations showing drug synergy in A549 and H23 cells. Using 11-point dose titrations and isobologram analysis of drug combinations, we see strong synergy among PI3 kinase, Raf and Erk inhibitors in these cells. Using the recently described Kras G12C inhibitor, expressed in H23 cells, we have validated that sgRNA deletion of the the key Kras effector for specific pathway,s including cell adhesion (RADIL), growth signaling (RAF) and endocytosis/ macropinocytosis (RIN), is affected and that use of the Kras inhibitor ARS-853 shows much-reduced effects on specific Kras effector pathways in cells deleted for these effectors. These systematic data underscore the limitations of our current understanding of Kras-driven cancers, revealing new genetic vulnerabilities and target candidates. Citation Format: Peter K. Jackson. A combined protein-protein interaction and genetic interaction map defines new and critical Kras effectors in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1313.

Abstract 4339: Molecular subtypes and novel genetic mechanisms of primary and acquired anti-EGFR resistance in colorectal cancer in the Prospect C biomarker trial

Abstract Despite stratification of only RAS wt CRCs for anti-EGFR antibody (aEa) therapy, many patients (pts) do not benefit and the molecular resistance (res) landscape remains incompletely understood. In order to decipher novel res mechanisms, we treated 40 RAS wt CRCs with single-agent aEa in a prospective trial and applied exome- and RNA-Seq to biopsies (bx) taken at baseline (BL) and at progression (PD). Among 20 BL bx from tumors with primary progression, 7 showed BRAF V600E mutations (mut), 3 harbored noncanonical BRAF D594K and/or KRAS L19F/A18D mut or KRAS amplifications (amp), 1 had a MEK1 mut and 1 was ERBB2 amp. Inactivating mut of the NF1 gene, a negative regulator of RAS, were found as novel mechanisms of primary res in 2 pts. No genetic drivers of primary res could be identified in 6/20 bx. Using transcriptomic analysis, 78% of pts with prolonged clinical benefit (≥16wks) displayed the Transit-Amplifying (TA) molecular subtype (Sadanandam et al., 2013). Stem-like (SL), goblet and inflammatory subtypes predominated (75%) among primary progressors. This significant enrichment in aEa sensitive CRCs (p=0.017) validates the TA subtype as a predictive biomarker. Exome- and deep Seq of PD bx from 13 pts who acquired res after prolonged clinical benefit detected KRAS mut or amp in only 2 cases. The FGFR2 ligand FGF10 was amplified in 1 PD bx and was validated in vitro as a novel mechanism of acquired res. No mut/amp of genes to which aEa res is usually attributed (RAS/RAF, EGFR, MET, ERBB2, MEK1) were found in the remaining 10 PD bx. Ultra-sensitive circulating tumor DNA-Seq at PD in 7 of these pts detected EGFR exodomain and RAS mut, including parallel evolution of multiple mut, in 3 cases. However, comparison to truncal TP53/APC mut showed that these res drivers were confined to small subclones and could therefore not explain the bulk of res. Overall, aberrations of RAS/RAF-pathway members and regulators were less abundant as drivers of acquired aEa res in this prospective trial than in reported retrospective series. Hence, we suspected additional novel mechanisms of acquired res. Molecular subtype switching from the aEa sensitive TA subtype to the comparatively insensitive SL subtype occurred in paired BL/PD bx from 3/7 pts who progressed after prolonged aEa benefit without detectable genetic res drivers in PD bx. Subtype switching was not observed in any of 6 analyzed BL/PD pairs from primary progressors. Considering the strong association of aEa sensitivity with TA subtype at BL, these data suggest subtype switching from TA to SL as a novel mechanism of acquired aEa res. This prospective trial revealed novel genetic (NF1 mut, FGF10 amp) and likely transcriptomic (TA to SL subtype switching) mechanisms of aEa resistance in CRC. These results should enable more precise aEa therapy stratification and may open opportunities to prevent res through SL subtype targeting strategies. Citation Format: Khurum Hayat Khan, Andrew Woolston, Georgia Spain, Louise Barber, Yatish Patil, Beatrice Griffiths, Reyes GonzalezExposito, Sonia Mansukhani, Matthew Davies, Sheela Rao, David Watkins, Francesco Sclafani, Jana Suntharanathan, Clare Peckitt, Ruwaida Begum, Isma Rana, Janet Thomas, Jacqui Oates, Annette Bryant, Andrew Wotherspoon, Nicos Fotiadis, Nasir Khan, Sebastian Guettler, Katharina von Loga, Naureen Starling, Ian Chau, Anguraj Sadanandam, David Cunningham, Marco Gerlinger. Molecular subtypes and novel genetic mechanisms of primary and acquired anti-EGFR resistance in colorectal cancer in the Prospect C biomarker trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4339.

Yeast and Cancer: Common Mechanism Underlying Activation of Ras by Glycolytic Flux

Ras proteins are small guanosine triphosphatases (GTPases), which function as on-off binary switches for diverse signaling pathways. Nearly 30% of human tumors have mutated forms of Ras. Both in the yeast Saccharomyces cerevisiae and in mammalian cells, Ras is an important regulator of cell proliferation. Mammalian Ras can functionally replace yeast Ras and hyperactive Ras oncogenes cause aberrant cell proliferation in yeast. Also the Ras regulatory proteins, guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), are homologous between yeast and mammalian cells. We used the tps1Δ mutant of the yeast S. cerevisiae as a tool to study Ras regulation. In the presence of glucose, this strain cannot grow due to a major metabolic deregulation causing hyperaccumulation of fructose-1,6-bisphosphate (Fru-1,6-bisP) and also hyperactivation of Ras leading to apoptosis. This led us to the finding that upon addition of glucose to wild type yeast cells, Fru-1,6-bisP promotes - in physiological concentrations - the activation of Ras through the GEF factor Cdc25. This activation may also be relevant in tumor biology because of the high glycolytic flux and higher concentrations of glycolytic intermediates present in cancer cells (Warburg effect). In vitro studies showed that Fru-1,6-bisP helps to increase the dissociation rate of the human K-Ras/SOS1 complex. Furthermore, we were able to show that glucose addition to glucose-deprived HEK293T and Hela Kyoto cells triggers rapid activation of Ras, as well as activation of its downstream targets MEK and ERK. Our results suggest that there may be a causal link between the high glycolytic activity and rapid cell proliferation of yeast and cancer cells in glucose medium through Fru-1,6-bisP activation of Ras. Support or Funding Information This work was supported by a PhD fellowship from the Agency for Innovation by Science and Technology (IWT-Flanders) to K.P., F.V.L., D.H., and W.V., by a post- doctoral F+-fellowship from the Research Fund of the KU Leuven to H.Q., and by grants from the Fund for Scientific Research—Flanders, Interuniversity Attraction Poles Net- work P6/14 and P7/40, and the Research Fund of the KU Leuven (Concerted Research Actions) to J.M.T., and a grant from the Belgian Foundation Against Cancer (FAF-F/ 2016/822) to V.J. Schematic overview of initial glucose metabolism in yeast and its connection to activation of the Ras-cAMP-PKA pathway. This pathway exerts major control over cell proliferation, fermentation rate, stress tolerance and developmental pathways in yeast. We show that Fru1,6bisP functions as activator of Ras by interacting with Cdc25 and that this connection between glycolysis and Ras is conserved between yeast and mammalian cells. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ERK1/2 inhibitors: New weapons to inhibit the RAS-regulated RAF-MEK1/2-ERK1/2 pathway.

The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is de-regulated in a variety of cancers due to mutations in receptor tyrosine kinases (RTKs), negative regulators of RAS (such as NF1) and core pathway components themselves (RAS, BRAF, CRAF, MEK1 or MEK2). This has driven the development of a variety of pharmaceutical agents to inhibit RAF-MEK1/2-ERK1/2 signalling in cancer and both RAF and MEK inhibitors are now approved and used in the clinic. There is now much interest in targeting at the level of ERK1/2 for a variety of reasons. First, since the pathway is linear from RAF-to-MEK-to-ERK then ERK1/2 are validated as targets per se. Second, innate resistance to RAF or MEK inhibitors involves relief of negative feedback and pathway re-activation with all signalling going through ERK1/2, validating the use of ERK inhibitors with RAF or MEK inhibitors as an up-front combination. Third, long-term acquired resistance to RAF or MEK inhibitors involves a variety of mechanisms (KRAS or BRAF amplification, MEK mutation, etc.) which re-instate ERK activity, validating the use of ERK inhibitors to forestall acquired resistance to RAF or MEK inhibitors. The first potent highly selective ERK1/2 inhibitors have now been developed and are entering clinical trials. They have one of three discrete mechanisms of action - catalytic, "dual mechanism" or covalent - which could have profound consequences for how cells respond and adapt. In this review we describe the validation of ERK1/2 as anti-cancer drug targets, consider the mechanism of action of new ERK1/2 inhibitors and how this may impact on their efficacy, anticipate factors that will determine how tumour cells respond and adapt to ERK1/2 inhibitors and consider ERK1/2 inhibitor drug combinations.

Photo Regulation of Small G-Protein RAS using Photochromic Peptide

The small guanine nucleotide binding proteins (G-protein), which are known as a molecular switch is central regulator of cellular signaling pathway. The regulation mechanism of G-protein is well studied at molecular level. Ras is one of the G-protein which have essential role in cellular signal is regulated by guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). GTP-Ras induced by GEF is active state, whereas GDP-Ras induced by GAP is inactive state. The conformational change induced by GTP binding enables Ras to transduce a signal into downstream through direct interaction with its effectors. Also, mutant Ras known as constitutive active cause excess signaling transduction to downstream effectors, which is pathogenesis of canceration. In this study, we focused on the interaction between Ras and its GEF, Son of Sevenless (SOS) to control Ras function using photochromic compounds. Previously it was shown that the peptide mimicking the αH-helix, Ras binding region of SOS effectively inhibits Ras function competing with native SOS. Therefore, it is expected that photo-induced structural change of the peptide conjugated with photochromic compound enable us to control Ras activity indirectly. We designed and synthesized several SOS αH-Helix peptides which contains two cysteine residues in order to cross-link with bi-functional photochromic molecule of azobenzene derivative, ABDM which change its structure reversibly by UV-VIS irradiation. CD and NMR analysis revealed that the modification of the peptide with ABDM stabilizes secondary structure and UV and VIS light irradiations induce secondary structural change reversibly. SOS dependent nucleotide exchange reaction of Ras in the presence of ABDM-SOS peptide were also altered photo-reversibly. Spontaneous cellular uptake of FITC conjugated peptide was observed by confocal microscopy. We also examined the photoreversible ERK pathway inhibition with ABDM-peptide in HeLa cell.

Characterisation of HRas local signal transduction networks using engineered site-specific exchange factors

ABSTRACT Ras GTPases convey signals from different types of membranes. At these locations, different Ras isoforms, interactors and regulators generate different biochemical signals and biological outputs. The study of Ras localisation-specific signal transduction networks has been hampered by our inability to specifically activate each of these Ras pools. Here, we describe a new set of site-specific tethered exchange factors, engineered by fusing the RasGRF1 CDC25 domain to sub-localisation-defining cues, whereby Ras pools at specific locations can be precisely activated. We show that the CDC25 domain has a high specificity for activating HRas but not NRas and KRas. This unexpected finding means that our constructs mainly activate endogenous HRas. Hence, their use enabled us to identify distinct pathways regulated by HRas in endomembranes and plasma membrane microdomains. Importantly, these new constructs unveil different patterns of HRas activity specified by their subcellular localisation. Overall, the targeted GEFs described herein constitute ideal tools for dissecting spatially-defined HRas biochemical and biological functions.

Concepts and advances in cancer therapeutic vulnerabilities in RAS membrane targeting.

For decades oncogenic RAS proteins were considered undruggable due to a lack of accessible binding pockets on the protein surfaces. Seminal early research in RAS biology uncovered the basic paradigm of post-translational isoprenylation of RAS polypeptides, typically with covalent attachment of a farnesyl group, leading to isoprenyl-mediated RAS anchorage at the plasma membrane and signal initiation at those sites. However, the failure of farnesyltransferase inhibitors to translate to the clinic stymied anti-RAS therapy development. Over the past ten years, a more complete picture has emerged of RAS protein maturation, intracellular trafficking, and location, positioning and retention in subdomains at the plasma membrane, with a corresponding expansion in our understanding of how these properties of RAS contribute to signal outputs. Each of these aspects of RAS regulation presents a potential vulnerability in RAS function that may be exploited for therapeutic targeting, and inhibitors have been identified or developed that interfere with RAS for nearly all of them. This review will summarize current understanding of RAS membrane targeting with a focus on highlighting development and outcomes of inhibitors at each step.

P1464Extreme RAS regulation in HFrEF already on optimal treatment: the low and high renin phenotype heart failure

P1464Extreme RAS regulation in HFrEF already on optimal treatment: the low and high renin phenotype heart failure

Abstract 1355: Linking AP/MS-derived protein-protein interaction networks and pairwise sgRNA screens defines new KRAS effectors in non-small cell lung cancer

Abstract The KRAS oncogene is mutated in over one fourth of human cancers. Despite decades of work studying known effector pathways, no drugs in clinical use specifically target KRAS-mutant tumors. Uncharacterized feedback mechanisms and parallel pathways have stymied the treatment of KRAS-mutant tumors in patients, and the KRas protein does not easily accommodate the binding of inhibitory small molecules. These challenges demand better characterization of the physical and genetic relationships between Ras regulators and effectors. To that end, we used tandem affinity purification of Kras, Hras and Nras and twelve proteins with known regulatory or effector roles to generate a high-confidence protein-protein interaction (PPI). Interactions were cross-referenced with public PPI, genetic susceptibility, and patient data to assemble a physical PPI map annotated with important functional determinants. The final map contains 287 genes and 990 physical interactions among them. This map was used to construct a library of >1000 sgRNAs covering >100 genes of probable relevance to the Ras pathway. This library was screened for pairwise genetic effects in a dual sgRNA vector system to identify quantitative changes in the growth of the A549 NSCLC line. We will present the quantitative results of this screen and additional validation studies on several previously undescribed regulators and effectors of the Ras proteins. In particular, we highlight unknown interactions between KRAS and the cell adhesion regulator RADIL, and the specificity of Ras-family protein interaction with the Raf and Ral GEF family proteins. These data underscore the limitations of our current understanding of Kras-driven cancers and reveal their genetic vulnerabilities. Citation Format: Marcus R. Kelly. Linking AP/MS-derived protein-protein interaction networks and pairwise sgRNA screens defines new KRAS effectors in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1355. doi:10.1158/1538-7445.AM2017-1355

GSK-3 as a novel prognostic indicator in leukemia.

While leukemias represent a diverse set of diseases with malignant cells derived from myeloid or lymphoid origin, a common feature is the dysregulation of signal transduction pathways that influence leukemogeneisis, promote drug resistance, and favor leukemia stem cells. Mutations in PI3K, PTEN, RAS, or other upstream regulators can activate the AKT kinase which has central roles in supporting cell proliferation and survival. A major target of AKT is Glycogen Synthase Kinase 3 (GSK3). GSK3 has two isoforms (alpha and beta) that were studied as regulators of metabolism but emerged as central players in cancer in the early 1990s. GSK3 is unique in that the isoforms are constitutively active. Active GSK3 promotes destruction of oncogenic proteins such as beta Catenin, c-MYC, and MCL-1 and thus has tumor suppressor properties. In AML, inactivation of GSK3 is associated with poor overall survival. Interestingly in some leukemias GSK3 targets a tumor suppressor and thus the kinases can act as tumor promoters in those instances. An example is GSK3 targeting p27Kip1 in AML with MLL translocation. This review will cover the role of GSK3 in various leukemias both as tumor suppressor and tumor promoter. We will also briefly cover current state of GSK3 inhibitors for leukemia therapy.

A Transposon-based Analysis Reveals RASA1 Is Involved in Triple-Negative Breast Cancer.

RAS genes are mutated in 20% of human tumors, but these mutations are very rare in breast cancer. Here, we used a mouse model to generate tumors upon activation of a mutagenic T2Onc2 transposon via expression of a transposase driven by the keratin K5 promoter in a p53+/- background. These animals mainly developed mammary tumors, most of which had transposon insertions in one of two RASGAP genes, neurofibromin1 (Nf1) and RAS p21 protein activator (Rasa1). Immunohistochemical analysis of a collection of human breast tumors confirmed that low expression of RASA1 is frequent in basal (triple-negative) and estrogen receptor negative tumors. Bioinformatic analysis of human breast tumors in The Cancer Genome Atlas database showed that although RASA1 mutations are rare, allelic loss is frequent, particularly in basal tumors (80%) and in association with TP53 mutation. Inactivation of RASA1 in MCF10A cells resulted in the appearance of a malignant phenotype in the context of mutated p53. Our results suggest that alterations in the Ras pathway due to the loss of negative regulators of RAS may be a common event in basal breast cancer. Cancer Res; 77(6); 1357-68. ©2017 AACR.

Aurora kinase A interacts with H-Ras and potentiates Ras-MAPK signaling.

In cancer, upregulated Ras promotes cellular transformation and proliferation in part through activation of oncogenic Ras-MAPK signaling. While directly inhibiting Ras has proven challenging, new insights into Ras regulation through protein-protein interactions may offer unique opportunities for therapeutic intervention. Here we report the identification and validation of Aurora kinase A (Aurora A) as a novel Ras binding protein. We demonstrate that the kinase domain of Aurora A mediates the interaction with the N-terminal domain of H-Ras. Further more, the interaction of Aurora A and H-Ras exists in a protein complex with Raf-1. We show that binding of H-Ras to Raf-1 and subsequent MAPK signaling is enhanced by Aurora A, and requires active H-Ras. Thus, the functional linkage between Aurora A and the H-Ras/Raf-1 protein complex may provide a mechanism for Aurora A's oncogenic activity through direct activation of the Ras/MAPK pathway.

Abstract A38: MTH1 Inhibition as an effective tumor-suppressive strategy in RAS-driven cancer

Abstract Oncogenic RAS mutations confer multiple malignant traits to cancer cells, many of which are mediated by oncogene-induced reactive oxygen species (ROS). However, oncogenic ROS also create vulnerabilities in the cancer cells, sensitizing them to oxidative DNA damage and strand breaks which can trigger cellular senescence or cell death. Thus RAS-driven tumor cells require adaptive redox protective mechanisms to inhibit ROS-associated tumor suppression. We previously reported that MutT Homolog 1 (MTH1), the mammalian 8-oxodGTPase, comprises one such critical adaptation. We have shown MTH1 is important for facilitating the full gamut of RAS-driven malignancy by promoting evasion of the first barrier to transformation, viz. oncogene-induced senescence (OIS), enhancing RAS-mediated transformation and related pro-malignant traits, and maintaining proliferation and tumorigenicity in established RAS-driven tumor cells. Recently chemical inhibitors against MTH1 have been developed, although there is some controversy surrounding their efficacy and mode of action. Our work here discusses RAS regulation of MTH1 expression, and the molecular contexts under which MTH1 inhibition can synergize with oncogenic oxidative stress in RAS-driven tumor cells for optimal tumor suppressive response. Citation Format: Govindi Jayanika Samaranayake, Clara Issabella Troccoli, Tyler Andrew Cunningham, Priyamvada Rai. MTH1 Inhibition as an effective tumor-suppressive strategy in RAS-driven cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A38.