CDC25 Domain Research Articles

A Focused Review of Ras Guanine Nucleotide-Releasing Protein 1 in Immune Cells and Cancer.

Four Ras guanine nucleotide-releasing proteins (RasGRP1 through 4) belong to the family of guanine nucleotide exchange factors (GEFs). RasGRPs catalyze the release of GDP from small GTPases Ras and Rap and facilitate their transition from an inactive GDP-bound to an active GTP-bound state. Thus, they regulate critical cellular responses via many downstream GTPase effectors. Similar to other RasGRPs, the catalytic module of RasGRP1 is composed of the Ras exchange motif (REM) and Cdc25 domain, and the EF hands and C1 domain contribute to its cellular localization and regulation. RasGRP1 can be activated by a diacylglycerol (DAG)-mediated membrane recruitment and protein kinase C (PKC)-mediated phosphorylation. RasGRP1 acts downstream of the T cell receptor (TCR), B cell receptors (BCR), and pre-TCR, and plays an important role in the thymocyte maturation and function of peripheral T cells, B cells, NK cells, mast cells, and neutrophils. The dysregulation of RasGRP1 is known to contribute to numerous disorders that range from autoimmune and inflammatory diseases and schizophrenia to neoplasia. Given its position at the crossroad of cell development, inflammation, and cancer, RASGRP1 has garnered interest from numerous disciplines. In this review, we outline the structure, function, and regulation of RasGRP1 and focus on the existing knowledge of the role of RasGRP1 in leukemia and other cancers.

Structural Insights into Phospholipase Cε

PLC (phospholipase C) enzymes hydrolyze phosphatidylinositol lipids to generate inositol phosphates (IPx) and diacylglycerol (DAG). These second messengers stimulate intracellular calcium release and protein kinase C activation. PLCε is the most recently identified member of the PLC family, and is activated downstream of G protein‐coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through direct interactions with G proteins. In the cardiovascular system, PLCε is necessary for normal cardiac contractility, and its dysregulation contributes to cardiac hypertrophy and heart failure. PLCε shares a highly conserved core that is observed in other PLC subfamilies, and contains a unique N‐terminal CDC25 domain and two C‐terminal Ras association (RA) domains. However, structural insights into the full‐length protein, or catalytically active fragments that contain two or more regulatory domains, have been lacking. Using a combination of structural and functional studies, we previously showed that the PH domain and first two EF hands are dynamic in solution, providing an explanation as to why high‐resolution structures have been elusive. We are now pursuing high‐resolution structural studies of larger PLCε variants and the full‐length enzyme using single particle cryo‐electron microscopy (cryo‐EM). To facilitate this process, we are utilizing domain‐specific fab antibodies as fiduciary markers and to stabilize this conformationally heterogeneous protein. Understanding the structure of PLCε will be critical in identifying sites that can be targeted by small molecule chemical probes, ultimately validating this enzyme as a therapeutic target in cardiovascular disease.

Autopromotion of K-Ras4B Feedback Activation Through an SOS-Mediated Long-Range Allosteric Effect.

The Ras-specific guanine nucleotide exchange factors Son of Sevenless (SOS) regulates Ras activation by converting inactive GDP-bound to active GTP-bound states. The catalytic activity of Ras is further allosterically regulated by GTP−Ras bound to a distal site through a positive feedback loop. To address the mechanism underlying the long-range allosteric activation of the catalytic K-Ras4B by an additional allosteric GTP–Ras through SOS, we employed molecular dynamics simulation of the K-Ras4BG13D•SOScat complex with and without an allosteric GTP-bound K-Ras4BG13D. We found that the binding of an allosteric GTP−K-Ras4BG13D enhanced the affinity between the catalytic K-Ras4BG13D and SOScat, forming a more stable conformational state. The peeling away of the switch I from the nucleotide binding site facilitated the dissociation of GDP, thereby contributing to the increased nucleotide exchange rate. The community networks further showed stronger edge connection upon allosteric GTP−K-Ras4BG13D binding, which represented an increased interaction between catalytic K-Ras4BG13D and SOScat. Moreover, GTP−K-Ras4BG13D binding transmitted allosteric signaling pathways though the Cdc25 domain of SOS that enhanced the allosteric regulatory from the K-Ras4BG13D allosteric site to the catalytic site. This study may provide an in-depth mechanism for abnormal activation and allosteric regulation of K-Ras4BG13D.

Calcium-Induced Conformational Changes in EF hands and Autoinhibitory Linker Region are Critical for Catalytic Activity of CalDAG-GEFI Towards the Small GTPase Rap1

IntroductionSignaling by the small GTPase Rap1B is critical for the rapid inside-out activation of platelet integrins, a prerequisite for platelet aggregation and hemostatic plug formation at sites of vascular injury. The guanine nucleotide exchange factor (GEF), CalDAG-GEFI (CDGI), is the predominant Rap-GEF in platelets. Cell biological studies demonstrated that agonist receptor-mediated increases in cytosolic calcium are necessary for CDGI activity, but it is currently not known if and how calcium affects CDGI activity.MethodsRecombinant CDGI (1-551) and Rap1B (1-181) proteins were purified to test nucleotide exchange activity in a fluorescence-based functional assay. Hydrogen-deuterium exchange mass spectrometry was utilized to measure dynamic differences between the active and inactive forms of CDGI.ResultsNucleotide exchange activity of CDGI was strongly diminished by substitution of calcium-binding residues E450A or E479A within EF hand 1 and EF hand 2, respectively. Addition of exogenous calcium restored catalytic activity in CDGI (E479A) but not CDGI (E450A) or CDGI (E450A+E479A). Calcium-bound active CDGI exhibited faster deuterium exchange compared to the calcium-free inactive form, in particular for residues within the EF hands and a potential autoinhibitory linker region connecting the EF hand domains and the Cdc25 catalytic domain.Consistent with regulation by an autoinhibitory linker, catalytic activity was fully restored in CDGI (E450A+E479A) by a gain-of-function substitution in valine 406.ConclusionsThese findings propose a model where calcium-induced conformational changes within the EF hands and autoinhibitory linker region are necessary for Rap1B to gain access to the catalytic binding pocket in the Cdc25 domain of CDGI. These findings will be important in our understanding of how mutations in patients impair CDGI function and in efforts to design inhibitors of CDGI signaling. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular Mechanism of Rap1A‐Dependent Activation of PLCɛ

Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce diacylglycerol (DAG) and inositol phosphates, leading to the activation of protein kinase C (PKC) and downstream signaling pathways, including cell growth and survival. The PLCɛ subfamily is a key player in cardiovascular function, where it contributes to maximum contractility. However, prolonged activation of PLCɛ results in cardiac hypertrophy and heart failure through its ability to regulate the expression of hypertrophic genes. This process is regulated by the small GTPase Rap1A, which is activated downstream of β‐adrenergic receptors. Rap1A binds to the C‐terminal Ras association (RA) domain of PLCɛ, simultaneously translocating the complex to the perinuclear region and activating PLCɛ. PLCɛ also contains an N‐terminal CDC25 domain, which has guanine nucleotide exchange factor (GEF) activity for Rap1A resulting in a feed forward activation loop and sustained lipid hydrolysis. However, the molecular mechanism of this process is not known. In this work, we seek to characterize the interactions between Rap1A and PLCɛ using structural and functional studies to map the Rap1A binding site on PLCɛ and determine whether activation results in conformational changes that release autoinhibition and/or increase membrane association. These studies provide the first molecular details of the Rap1A‐dependent activation of PLCɛ and open the door to the development of new therapeutic strategies for treating cardiac hypertrophy.Support or Funding InformationThis research is supported by the American Heart Association Predoctoral Fellowship 18PRE33990057 to M.S., American Heart Association Grant Scientist Development Grant 16SDG29920017 to A.M.L. and the NIH NLHBI 1R01HL 141076‐01 to A.M.L. SAXS data was collected and analyzed with the assistance of S. Chakravarthy at Argonne National Laboratory.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural Insights into Phospholipase Cɛ Function

Phospholipase Cɛ (PLCɛ) is a member of the PLC family of enzymes that hydrolyze phosphatidylinositol lipids downstream of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). PLCɛ is unique among the PLC superfamily as it contains an N‐terminal CDC25 domain, which has guanine nucleotide exchange factor (GEF) activity towards small G proteins Rap1A, and two C‐terminal Ras association (RA) domains. The best characterized pathway leading to PLCɛ activation is mediated through β‐adrenergic receptor (βARs) signaling. Stimulation of these receptors culminates in the activation of the small GTPase, Rap1A, which binds directly to PLCɛ and translocates the complex to the Golgi. There, PLCɛ hydrolyzes phosphatidylinol‐4‐phosphate (PI4P). Prolonged activation of this pathway results in increased expression of hypertrophy‐related genes. However, how the structural basis of PLCɛ activity under basal conditions and upon activation by Rap1A are unknown. Towards this end, we have obtained the first high‐resolution insights into a catalytically active PLCɛ variant. These studies, together with biochemical analysis of the intramolecular contacts observed in the structure, provide the first molecular insights into this enzyme.Support or Funding InformationThis research is supported by American Heart Association Scientist Development Grant 16SDG29920017 and NIH 1R01HL141076‐01 to A.M.LThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Allosteric KRas4B Can Modulate SOS1 Fast and Slow Ras Activation Cycles

Membrane-anchored Ras family proteins are activated by guanine nucleotide exchange factors such as SOS1. The CDC25 domain of SOS1 catalyzes GDP-to-GTP exchange, thereby activating Ras. Here, we aim to decipher the activation mechanism of KRas4B, a significantly mutated oncogene. We perform large-scale molecular dynamics simulations on 12 SOS1 systems, scrutinizing each step in two possible KRas4B activation cycles, fast and slow. To activate KRas4B at the CDC25 catalytic site, the allosteric site in the Ras exchanger motif (REM) domain of SOS1 needs to recruit a (nucleotide-bound) KRas4B molecule. Our simulations indicate that KRas4B-GTP interacts with the REM allosteric site more strongly than with the CDC25 catalytic site, consistent with its allosteric role in the GDP-to-GTP exchange. In the fast cycle, the allosteric KRas4B-GTP induces conformational change at the catalytic site. The conformational change facilitates loading KRas4B-GDP at the catalytic site and opening the KRas4B nucleotide-binding site for GDP release and GTP binding. GTP binding reduces the affinity of KRas4B-GTP to the CDC25 catalytic site, resulting in its release. By contrast, in the slow cycle, KRas4B-GDP binds at the allosteric REM site. The limited, altered conformational change that it induces prevents the exact alignments of switch I and II of KRas4B. The increasing binding strength at both binding sites due to interactions of regions other than switch I and II retards GDP release from the catalytic KRas4B, thus KRas4B activation. The accelerated activation cycle supports a positive feedback loop with allosteric signals communicating between the two Ras molecules and is the predominant, native function of SOS. SOS1 activation details may help drug discovery to inhibit Ras activation.

Abstract 683: SOS1 allosterically linked conformational changes modulate KRas4B activation

Abstract Son of sevenless homology 1 (SOS1) is a robust guanine nucleotide exchange factor (GEF) that exchanges GDP by GTP and activates Ras. SOS1 is a large multidomain protein with molecular weight of ~153 kDa. The C-terminal catalytic region, composed of RAS exchanger motif (REM) and CDC25 domains, is responsible for Ras activation. SOS1 offers two Ras binding sites; one at the allosteric site in REM and the other at the catalytic site in CDC25. Experimental studies provided a Ras-SOS1-Ras ternary complex and suggested that Ras binding to REM allosteric site is essential to activate GDP-bound Ras at the CDC25 catalytic site. However, details of SOS1 conformational changes due to different binding modes of Ras at the allosteric and catalytic sites in Ras activation are not well understood. Here, using molecular dynamics (MD) simulations, we examine Ras activation mechanism for SOS1 systems containing GDP- and GTP-bound, and nucleotide-free KRas4B. Among the three Ras isoforms, including HRas, NRas, and KRas (with two splice variants of KRas4A and KRas4B), KRas4B is highly oncogenic, the most frequently mutated in RAS-driven cancers. We observed that the REM domain inherently and sterically obstructs the Ras binding site of CDC25 domain. Upon binding to the REM allosteric site, KRas4B-GTP relieves the steric occlusion, making the CDC25 catalytic site capable of associating with KRas4B-GDP, leading to the nucleotide exchange. Allosteric effects lead to the dislocation of the helix-hairpin motif at the CDC25 catalytic site. This large movement of the helix-hairpin motif causes the Ras catalytic site to open and liberates GDP. Allosteric signals propagating through the REM-CDC25 tandem domains of SOS1 can explain the communication between two Ras proteins modulating the large conformational changes. Our simulations provide insight into the structural correlation of KRas4B-SOS1 as well as the allosteric effect. The conformational changes of SOS1 correspond to the allosteric signaling between two KRas4B at REM and CDC25 domains, promoting Ras activation. Citation Format: Tsung-Jen Liao, Hyunbum Jang, David Fushman, Ruth Nussinov. SOS1 allosterically linked conformational changes modulate KRas4B activation [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 683.

Calcium-induced structural rearrangements release autoinhibition in the Rap-GEF CalDAG-GEFI

Platelets are recruited to sites of vascular injury, where they are activated and aggregate to form a hemostatic plug. This process requires the activation of the small GTPase Rap1B by its cognate guanine nucleotide exchange factor CalDAG-GEFI. Studies on platelet function suggest that CalDAG-GEFI activity is regulated by changes in cytosolic calcium, but the exact molecular mechanism is poorly understood. Here we show that purified CalDAG-GEFI is autoinhibited and directly regulated by calcium. Substitutions of putative calcium-binding residues within the canonical EF hands of CalDAG-GEFI diminish its capacity to activate Rap1B. Structural differences between active (WT) and inactive (EF hand variant) CalDAG-GEFI protein were determined by hydrogen-deuterium exchange MS. The highest differential rates of deuterium uptake in WT over EF hand variant CalDAG-GEFI were observed in regions within the catalytic Cdc25 domain and a putative autoinhibitory linker connecting the Cdc25 and EF hand domains. Exchange activity in the EF hand variant was fully restored by an additional substitution, valine 406 to glutamate, which is thought to disrupt the interface between the autoinhibitory linker and the Cdc25 domain. Overall, our results suggest a model for how CalDAG-GEFI remains in an autoinhibited state when levels of cytosolic calcium in resting platelets are low. In response to cellular stimulation, calcium mobilization and binding to the EF hands causes conformational rearrangements within CalDAG-GEFI, including the autoinhibitory linker that frees the catalytic surface of CalDAG-GEFI to engage and activate Rap1B. The data from this study are the first evidence linking CalDAG-GEFI activity directly to calcium.

G protein βγ subunits directly interact with and activate phospholipase CΕ

Phospholipase C (PLC) enzymes hydrolyze membrane phosphatidylinositol 4,5 bisphosphate (PIP2) and regulate Ca2+ and protein kinase signaling in virtually all mammalian cell types. Chronic activation of the PLCϵ isoform downstream of G protein-coupled receptors (GPCRs) contributes to the development of cardiac hypertrophy. We have previously shown that PLCϵ-catalyzed hydrolysis of Golgi-associated phosphatidylinositol 4-phosphate (PI4P) in cardiac myocytes depends on G protein βγ subunits released upon stimulation with endothelin-1. PLCϵ binds and is directly activated by Ras family small GTPases, but whether they directly interact with Gβγ has not been demonstrated. To identify PLCϵ domains that interact with Gβγ, here we designed various single substitutions and truncations of WT PLCϵ and tested them for activation by Gβγ in transfected COS-7 cells. Deletion of only a single domain in PLCϵ was not sufficient to completely block its activation by Gβγ, but blocked activation by Ras. Simultaneous deletion of the C-terminal RA2 domain and the N-terminal CDC25 and cysteine-rich domains completely abrogated PLCϵ activation by Gβγ, but activation by the GTPase Rho was retained. In vitro reconstitution experiments further revealed that purified Gβγ directly interacts with a purified fragment of PLCϵ (PLCϵ-PH-RA2) and increases PIP2 hydrolysis. Deletion of the RA2 domain decreased Gβγ binding and eliminated Gβγ stimulation of PIP2 hydrolysis. These results provide first evidence that Gβγ directly interacts with PLCϵ and yield insights into the mechanism by which βγ subunits activate PLCϵ.

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.

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.

Expanded repertoire of RASGRP2 variants responsible for platelet dysfunction and severe bleeding

AbstractHeritable platelet function disorders (PFDs) are genetically heterogeneous and poorly characterized. Pathogenic variants in RASGRP2, which encodes calcium and diacylglycerol-regulated guanine exchange factor I (CalDAG-GEFI), have been reported previously in 3 pedigrees with bleeding and reduced platelet aggregation responses. To better define the phenotype associated with pathogenic RASGRP2 variants, we compared high-throughput sequencing and phenotype data from 2042 cases in pedigrees with unexplained bleeding or platelet disorders to data from 5422 controls. Eleven cases harbored 11 different, previously unreported RASGRP2 variants that were biallelic and likely pathogenic. The variants included 5 high-impact variants predicted to prevent CalDAG-GEFI expression and 6 missense variants affecting the CalDAG-GEFI CDC25 domain, which mediates Rap1 activation during platelet inside-out αIIbβ3 signaling. Cases with biallelic RASGRP2 variants had abnormal mucocutaneous, surgical, and dental bleeding from childhood, requiring ≥1 blood or platelet transfusion in 78% of cases. Platelets displayed reduced aggregation in response to adenosine 5′-diphosphate and epinephrine, but variable aggregation defects with other agonists. There were no other consistent clinical or laboratory features. These data enable definition of human CalDAG-GEFI deficiency as a nonsyndromic, recessive PFD associated with a moderate or severe bleeding phenotype and complex defects in platelet aggregation.

PLCε1 regulates SDF-1α–induced lymphocyte adhesion and migration to sites of inflammation

Regulation of integrins is critical for lymphocyte adhesion to endothelium and migration throughout the body. Inside-out signaling to integrins is mediated by the small GTPase Ras-proximate-1 (Rap1). Using an RNA-mediated interference screen, we identified phospholipase Cε 1 (PLCε1) as a crucial regulator of stromal cell-derived factor 1 alpha (SDF-1α)-induced Rap1 activation. We have shown that SDF-1α-induced activation of Rap1 is transient in comparison with the sustained level following cross-linking of the antigen receptor. We identified that PLCε1 was necessary for SDF-1α-induced adhesion using shear stress, cell morphology alterations, and crawling on intercellular adhesion molecule 1 (ICAM-1)-expressing cells. Structure-function experiments to separate the dual-enzymatic function of PLCε1 uncover necessary contributions of the CDC25, Pleckstrin homology, and Ras-associating domains, but not phospholipase activity, to this pathway. In the mouse model of delayed type hypersensitivity, we have shown an essential role for PLCε1 in T-cell migration to inflamed skin, but not for cytokine secretion and proliferation in regional lymph nodes. Our results reveal a signaling pathway where SDF-1α induces T-cell adhesion through activation of PLCε1, suggesting that PLCε1 is a specific potential target in treating conditions involving migration of T cells to inflamed organs.

Thrombin Promotes Sustained Signaling and Inflammatory Gene Expression through the CDC25 and Ras-associating Domains of Phospholipase Cϵ

Phospholipase C-epsilon (PLCϵ) plays a critical role in G-protein-coupled receptor-mediated inflammation. In addition to its ability to generate the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, PLCϵ, unlike the other phospholipase C family members, is activated in a sustained manner. We hypothesized that the ability of PLCϵ to function as a guanine nucleotide exchange factor (GEF) for Rap1 supports sustained downstream signaling via feedback of Rap1 to the enzyme Ras-associating (RA2) domain. Using gene deletion and adenoviral rescue, we demonstrate that both the GEF (CDC25 homology domain) and RA2 domains of PLCϵ are required for long term protein kinase D (PKD) activation and subsequent induction of inflammatory genes. PLCϵ localization is largely intracellular and its compartmentalization could contribute to its sustained activation. Here we show that localization of PLCϵ to the Golgi is required for activation of PKD in this compartment as well as for subsequent induction of inflammatory genes. These data provide a molecular mechanism by which PLCϵ mediates sustained signaling and by which astrocytes mediate pathophysiological inflammatory responses.

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

Abstract IA17: 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 IA17. doi: 10.1158/1557-3125.RASONC14-IA17

Abstract 4417: Targeting Ras with small molecules that activate SOS-mediated nucleotide exchange

Abstract Aberrant activation of Ras, by mutation or constitutively active upstream kinases, occurs in nearly 30% of all human cancers, rendering Ras one of the most validated targets in cancer drug discovery. Historically, the direct inhibition of Ras with small molecules has proven extremely difficult. Here we report the discovery of compounds that bind to a unique pocket on the Ras:SOS:Ras complex, which increase the rate of SOScat-catalyzed nucleotide exchange in vitro and modulate Ras signaling pathways in cells. X-ray crystallography reveals that the molecules bind in a hydrophobic pocket in the CDC25 domain of SOS, adjacent to the Switch II region of Ras. Structure-based mutational analyses confirmed the functional relevance of this binding site and demonstrated that it is essential for compound activity. Consistent with their increased exchange activity in vitro, these molecules increase Ras-GTP levels in cells. However, the compounds inhibit ERK and AKT phosphorylation; the ERK inhibition at high concentrations is accompanied by an increase of p-ERK levels at lower compound concentrations. These molecules represent a unique tool to study the acute activation of Ras and highlight a novel pocket on SOS that may be exploited to modulate Ras signaling. Citation Format: Olivia W. Rossanese, Michael C. Burns, Qi Sun, R. Nathan Daniels, DeMarco V. Camper, J. Phillip Kennedy, Jason Phan, Edward T. Olejniczak, Taekyu Lee, Alex G. Waterson, Stephen W. Fesik. Targeting Ras with small molecules that activate SOS-mediated nucleotide exchange. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4417. doi:10.1158/1538-7445.AM2014-4417

Domain Contributions to Signaling Specificity Differences Between Ras-Guanine Nucleotide Releasing Factor (Ras-GRF) 1 and Ras-GRF2

Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2) constitute a family of similar calcium sensors that regulate synaptic plasticity. They are both guanine exchange factors that contain a very similar set of functional domains, including N-terminal pleckstrin homology, coiled-coil, and calmodulin-binding IQ domains and C-terminal Dbl homology Rac-activating domains, Ras-exchange motifs, and CDC25 Ras-activating domains. Nevertheless, they regulate different forms of synaptic plasticity. Although both GRF proteins transduce calcium signals emanating from NMDA-type glutamate receptors in the CA1 region of the hippocampus, GRF1 promotes LTD, whereas GRF2 promotes θ-burst stimulation-induced LTP (TBS-LTP). GRF1 can also mediate high frequency stimulation-induced LTP (HFS-LTP) in mice over 2-months of age, which involves calcium-permeable AMPA-type glutamate receptors. To add to our understanding of how proteins with similar domains can have different functions, WT and various chimeras between GRF1 and GRF2 proteins were tested for their abilities to reconstitute defective LTP and/or LTD in the CA1 hippocampus of Grf1/Grf2 double knock-out mice. These studies revealed a critical role for the GRF2 CDC25 domain in the induction of TBS-LTP by GRF proteins. In contrast, the N-terminal pleckstrin homology and/or coiled-coil domains of GRF1 are key to the induction of HFS-LTP by GRF proteins. Finally, the IQ motif of GRF1 determines whether a GRF protein can induce LTD. Overall, these findings show that for the three forms of synaptic plasticity that are regulated by GRF proteins in the CA1 hippocampus, specificity is encoded in only one or two domains, and a different set of domains for each form of synaptic plasticity.

Abstract PR01: Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

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 Information: Mol Cancer Ther 2013;12(11 Suppl):PR01. Citation Format: Michael Burns, Qi Sun, Richard Daniels, J. Phillip Kennedy, DeMarco Camper, Jason Phan, Edward Olejniczak, Taekyu Lee, Alex Waterson, Olivia Rossanese, Stephen Fesik. Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR01.