biomolecules Review The Ins and Outs of RAS Effector Complexes Christina Kiel 1,2, David Matallanas 1 and Walter Kolch 1,3,* 1 Systems Biology Ireland, School of Medicine, University College Dublin, Dublin 4, Ireland; [email protected] (C.K.); [email protected] (D.M.) 2 UCD Charles Institute of Dermatology, School of Medicine, University College Dublin, Dublin 4, Ireland 3 Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Dublin 4, Ireland * Correspondence: [email protected]; Tel.: +353-1-716-6303 Abstract: RAS oncogenes are among the most commonly mutated proteins in human cancers. They regulate a wide range of effector pathways that control cell proliferation, survival, differen- tiation, migration and metabolic status. Including aberrations in these pathways, RAS-dependent signaling is altered in more than half of human cancers. Targeting mutant RAS proteins and their downstream oncogenic signaling pathways has been elusive. However, recent results comprising detailed molecular studies, large scale omics studies and computational modeling have painted a new and more comprehensive portrait of RAS signaling that helps us to understand the intricacies of RAS, how its physiological and pathophysiological functions are regulated, and how we can target them. Here, we review these efforts particularly trying to relate the detailed mechanistic studies with global functional studies. We highlight the importance of computational modeling and data integration to derive an actionable understanding of RAS signaling that will allow us to design new mechanism-based therapies for RAS mutated cancers. Keywords: RAS oncogene; RAS signaling networks; RAS in human cancer; targeting RAS; computa- tional modeling; personalized therapies Citation: Kiel, C.; Matallanas, D.; Kolch, W. The Ins and Outs of RAS Effector Complexes. Biomolecules 2021, 1. Introduction 11, 236. https://doi.org/10.3390/ Since the discovery of retroviruses transducing RAS oncogenes in 1964 [1], huge re- biom11020236 search efforts have been spent on understanding what RAS does [2]. There is a good reason for that. RAS proteins are mutated in 19% of all human cancers, and several prevalent and Academic Editor: Daniel Abankwa deadly cancers, such as colorectal, lung, and pancreatic cancers and metastatic melanoma Received: 10 January 2021 are driven by RAS mutations [3]. Of the three RAS family members, KRAS is the most Accepted: 3 February 2021 frequently mutated form in pancreatic, colorectal, and lung cancer. NRAS is preferentially Published: 7 February 2021 mutated in melanoma and acute myeloid leukaemias, while HRAS mutations are rare [3]. In addition, germline mutations that activate RAS are responsible for RASopathies, a group Publisher’s Note: MDPI stays neutral of rare developmental disorders [4,5]. The oncogenic RAS mutations compromise the with regard to jurisdictional claims in ability of RAS proteins to hydrolyze guanosine triphosphate (GTP) to guanosine diphos- published maps and institutional affil- phate (GDP) or enhance the replacement of GDP by GTP. Physiologically, GTP binding iations. is stimulated by Guanine nucleotide Exchange Factors (GEFs), while GTP hydrolysis is accelerated by GTPase Activating Proteins (GAPs). In the GTP bound form, RAS switches into a conformation where it can bind and activate effector proteins which transduce a wide range of signals that regulate almost any aspect of cellular physiology [6]. The known Copyright: © 2021 by the authors. and well-characterized RAS signaling network is shown in Figure1. Upon GTP hydroly- Licensee MDPI, Basel, Switzerland. sis, RAS proteins release their effectors, thereby terminating the signal. Most often RAS This article is an open access article proteins are intermediates between surface receptors and intracellular signaling pathways distributed under the terms and that regulate proliferation, differentiation, survival, cell-cell interactions and migration [6]. conditions of the Creative Commons The oncogenic mutations are thought to lock RAS in the active conformation, causing Attribution (CC BY) license (https:// constitutive signaling through these downstream effector pathways, most prominently creativecommons.org/licenses/by/ the RAF and phosphoinositide-3 kinase (PI3K) pathways [6]. In fact, when one includes 4.0/). Biomolecules 2021, 11, 236. https://doi.org/10.3390/biom11020236 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, x FOR PEER REVIEW 2 of 29 Biomolecules 2021, 11, 236 2 of 28 and migration [6]. The oncogenic mutations are thought to lock RAS in the active confor- mation, causing constitutive signaling through these downstream effector pathways, most prominently the RAF and phosphoinositide-3 kinase (PI3K) pathways [6]. In fact, when onemutations includes in thesemutations two RASin these effector two pathways,RAS effector oncogenic pathways, activation oncogenic of the activation RAS pathway of the RASmay bepathway involved may in be more involved than half in more of all humanthan half cancers of all human [7]. Efforts cancers to target [7]. Efforts oncogenic to target RAS oncogenicsignaling have RAS beensignaling thwarted have by been the thwarted intricacies by of the RAS intricacies regulation of andRAS an regulation ever expanding and an evernetwork expanding of effectors network that of participate effectors inthat mediating participate its in oncogenic mediating effects its oncogenic [8]. The take-home effects [8]. Thelesson take was-home that welesson need was to understandthat we need RAS to signaling understand in much RAS greatersignaling detail, in much if we wantgreater to detail,successfully if we targetwant it.to Insuccessfully this review, target we summarize it. In this thereview, current we knowledgesummarize ofthe how current RAS knowledgeinteracts with of how its effectors RAS interacts focusing with on its modalities effectors focus that affecting on direct modalities interactions that affect as well direct as interactionsRAS isoform as and well tissue-specific as RAS isoform interactions. and tissue-specific interactions. Figure 1.1. Known RASRAS effectorseffectors and and signaling signaling pathways. pathways. The The RAS RAS activation activation cycle cycle comprises comprises GTP GTP binding binding aided aided by Guanineby Gua- nucleotidenine nucleotide Exchange Exchange Factors Factors (GEFs), (GEFs), which which puts RAS puts proteins RAS proteins into an into active an conformationactive conformation where itwhere can bind it can to bind effectors to effec- and activatetors and downstream activate downstream signaling signaling pathways. pathways. The main The and currentlymain and best-characterizedcurrently best-characterized RAS signaling RAS pathways signaling arepathways shown. Effectorsare shown. are Effectors released whenare released GTP is hydrolyzedwhen GTP is to hydrolyzed GDP, and this to intrinsic,GDP, and but this low, intrinsic, catalytic but RAS low activity, catalytic is vastly RAS accelerated activity is byvastly GTPase accelerated Activating by GTPase Proteins Activating (GAPs). Proteins (GAPs). 2. RAS Interactions with Effector Proteins 2.1. RAS Effector Proteins Bind to the ‘Switch‘Switch Regions’ of RASRAS∙GTP·GTP RAS effectors are specific specific for the GTP GTP-bound-bound conformation of RAS, where two struc- tural regions regions—the—the so so called called ‘switch ‘switch regions’ regions’ (switch (switch I and I and switch switch II)— II)—areare in a ‘closed’ in a ‘closed’ con- formation,conformation, which which exposes exposes interface interface residues residues involved involved in binding in binding to the to RB theD [9 RBD–12] [. 9Once–12]. theOnce γ phosphate the γ phosphate is hydrolyzed, is hydrolyzed, RAS∙GDP RAS acts·GDP like acts a ‘loaded like a ‘loadedspring’ and spring’ releases and releasesswitch I andswitch II in I and‘open’ II in conformations. ‘open’ conformations. While the While molecular the molecular switch function switch function of RAS ofbuilds RAS buildson the efficienton the efficient interaction interaction of effectors of effectors in the inGTP the-bound GTP-bound state, an state, interaction an interaction with the with GDP the- boundGDP-bound form is form not isstrictly not strictly impossible. impossible. Indeed, Indeed, through through structure structure-based-based mutagenesis mutagenesis of the CRAFof the- CRAF-RASRAS binding binding Domain Domain (RBD), (RBD),it was possible it was possible to engineer to engineer mutants mutants with high with affinity high foraffinity RAS∙GDP for RAS that·GDP induced that induced CRAF-mediated CRAF-mediated pathway pathway activity activity[13]. Importantly, [13]. Importantly, the 3D structurethe 3D structure of such of a suchhigh- aaffinity high-affinity RAS∙GDP RAS-·effectorGDP-effector complex complex showed showed a similar a similar closed closed con- formationconformation of the of switch the switch I/II regions, I/II regions, suggesting suggesting that the that conformation the conformation of RAS of in RAS that inregion that ratherregion than rather the than nucleotide the nucleotide state itself state defines itself defines the ability the ability to interact to interact with effector with effector [14]. [14]. Biomolecules 2021,, 11,, x 236 FOR PEER REVIEW 33 of of 29 28 2.2.2.2. Competition Competition for for Binding Binding to to RAS∙GTP RAS·GTP Generates Generates Different Different RAS RAS-Effector-Effector Complexes Complexes RASRAS effectors effectors are are defined defined