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Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling

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Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling G C A T T A C G G C A T genes Review Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling Aoife A. Nolan 1, Nourhan K. Aboud 1, Walter Kolch 1,2,* and David Matallanas 1,* 1 Systems Biology Ireland, School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland; [email protected] (A.A.N.); [email protected] (N.K.A.) 2 Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland * Correspondence: [email protected] (W.K.); [email protected] (D.M.) Abstract: Oncogenic RAS (Rat sarcoma) mutations drive more than half of human cancers, and RAS inhibition is the holy grail of oncology. Thirty years of relentless efforts and harsh disappointments have taught us about the intricacies of oncogenic RAS signalling that allow us to now get a pharma- cological grip on this elusive protein. The inhibition of effector pathways, such as the RAF-MEK-ERK pathway, has largely proven disappointing. Thus far, most of these efforts were aimed at blocking the activation of ERK. Here, we discuss RAF-dependent pathways that are regulated through RAF functions independent of catalytic activity and their potential role as targets to block oncogenic RAS signalling. We focus on the now well documented roles of RAF kinase-independent functions in apoptosis, cell cycle progression and cell migration. Keywords: RAF kinase-independent; RAS; MST2; ASK; PLK; RHO-α; apoptosis; cell cycle; cancer therapy Citation: Nolan, A.A.; Aboud, N.K.; Kolch, W.; Matallanas, D. Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling. 1. Introduction Genes 2021, 12, 553. https://doi.org/ RAS (Rat sarcoma) proteins are mutated in ca. 20% of all human cancers, with 10.3390/genes12040553 prevalent and deadly cancers such as colorectal, lung and pancreatic cancer featuring 40%, 20–40%, and >90% RAS mutations [1]. RAS proteins often have been described as Academic Editor: molecular switches that cycle between GDP-bound off and GTP-bound on states. When Esther Castellano-Sánchez switched on by the (normally receptor-induced) exchange of GDP versus GTP, they can bind to an array of >20 different types of effector proteins which mediate the downstream Received: 21 March 2021 biochemical and biological effects of RAS [2,3]. Oncogenic mutations keep RAS proteins in Accepted: 6 April 2021 Published: 10 April 2021 the GTP bound state resulting in the constitutive activation of pathways that stimulate cell proliferation, survival, invasiveness and drug resistance. Thus, inhibiting RAS has been an Publisher’s Note: MDPI stays neutral early aim for the development of targeted therapies for cancer [4]. with regard to jurisdictional claims in When efforts to inhibit RAS directly failed, the attention turned to the inhibition of published maps and institutional affil- downstream effector pathways. A main effector of oncogenic RAS signalling is the RAF- iations. MEK-ERK pathway (Figure1). This pathway is a cascade of three kinases. The first, RAF (Rapid Accelerated Fibrosarcoma), binds to GTP-loaded RAS and is a direct RAS effector. RAS-activated RAF phosphorylates and activates MEK (Dual specificity mitogen-activated protein kinase kinase 1), which in turn phosphorylates and activates ERK (extracellular signal-regulated kinase) [5]. RAF is a family of three kinases: RAF1, BRAF, and ARAF. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. The RAF-MEK-ERK pathway drives several features of oncogenic transformation, and This article is an open access article BRAF is an oncogene in its own right that is frequently mutated in melanoma, colorectal distributed under the terms and cancer, and lung cancer, amongst others [5,6]. Thus, drugs targeting the RAF-MEK-ERK conditions of the Creative Commons pathway seemed a logical and promising strategy. Potent RAF and MEK inhibitors were Attribution (CC BY) license (https:// developed and several are used in the clinic [6]. However, while effective against some creativecommons.org/licenses/by/ mutant BRAF-driven cancers, such as melanoma, they proved ineffective against RAS- 4.0/). mutated cancers [4,6]. A main reason is that RAS induces homo- and heterodimerization Genes 2021, 12, 553. https://doi.org/10.3390/genes12040553 https://www.mdpi.com/journal/genes Genes 2021, 12, x FOR PEER REVIEW 2 of 16 Genes 2021, 12, 553 2 of 16 RAS-mutated cancers [4,6]. A main reason is that RAS induces homo- and heterodimeri- ofzation RAF kinases,of RAF kinases, and that and the dimerthat the is resistantdimer is toresistant drug inhibition. to drug inhibition. The drug resistanceThe drug isre- causedsistance by is the caused ability by of the a drug-bound, ability of a drug-bound, catalytically inhibitedcatalytically RAF inhibited protomer RAF to allosterically protomer to transactivateallosterically thetransactivate kinase activity the kinase of the activity other protomer of the other [7, 8protomer]. This mechanism [7,8]. This mechanism leads to a paradoxicalleads to a paradoxical activation ofactivation ERK in RAS-mutated of ERK in RAS-mutated cells in response cells in to response RAF inhibitors to RAF[ 9inhibi-–11]. Wetors have [9–11]. recently We have shown recently that thisshown impasse that this can impasse be broken can by be combining broken by RAFcombining inhibitors, RAF choseninhibitors, by a sophisticatedchosen by a sophisticated computational computational model, that will model, effectively that will block effectively ERK activation block ERK in mutantactivation RAS in cells mutant [12]. RAS Other cells pharmacological [12]. Other pharmacological approaches to overcome approach RAFes to dimer-induced overcome RAF resistancedimer-induced to RAF resistance inhibitors to are RAF being inhibitors pursued are as wellbeing [13 pursued–15]. All as these well approaches [13–15]. All focus these onapproaches preventing focus the reactivationon preventing of ERKthe reactivation signalling. of ERK signalling. FigureFigure 1. 1.The The RAF-MEK-ERK RAF-MEK-ERK pathwaypathway isis activatedactivated byby H/KH/K and NRAS NRAS upon upon extracellular extracellular stimuli. stimuli. ERK1/2ERK1/2 phosphorylatephosphorylate overover 5050 substratessubstrates andand controlcontrol differentdifferent cell cell fate. fate. InIn this this review, review, we we focus focus on on a a complementary complementary arm arm of of RAF RAF functions, functions, which which is is the the controlcontrol of of signalling signalling pathways pathways independent independent of RAFof RAF kinase kinase catalytic catalytic functions. functions. RAF kinasesRAF ki- havenases several have several kinase-independent kinase-independent functions, function whichs, are which relevant are forrelevant cancer for development cancer develop- and progression.ment and progression. Here, we summarize Here, we summariz how thesee kinase-independent how these kinase-independent RAF functions RAF contribute functions tocontribute cancer and to explorecancer and how explor they coulde how be they targeted. could be targeted. 2.2. RAF RAF Interacting Interacting Proteins Proteins Regulated Regulated in in a a Kinase-Independent Kinase-Independent Fashion Fashion TheThe only only commonly commonly accepted accepted substrates substrates for for RAF RAF kinases kinases are are the the MEK1/2 MEK1/2 kinases, kinases, whosewhose only only known known substrates substrates are are ERK1/2. ERK1/2. ActivationActivation of of this this pathway pathway through through oncogenic oncogenic mutationsmutations ofof RAS,RAS, RAF or or MEK MEK kinases kinases can can drive drive cancer cancer cell cell proliferation. proliferation. However, However, sev- severaleral lines lines of evidence of evidence now now strongly strongly support support a contribution a contribution of kinase of kinase - independent - independent func- functionstions to the to theoncogenic oncogenic capacity capacity of this of this pathwa pathway.y. This This was was first first rea realizedlized in in2001, 2001, when when re- reportsports were were published thatthat knockingknocking out out the theRAF1 RAF1gene gene in micein mice causes causes apoptosis apoptosis indepen- inde- dentlypendently of its of kinase its kinase function function [16,17 [16,17].]. Mikula Mikula et al. et showed al. showed that that knocking knocking out RAF1out RAF1had had no impactno impact on the on activation the activation of the of ERK the pathway ERK pathway but resulted but resulted in increased in increased apoptosis apoptosis mainly inmainly the liver in the and liver haematopoietic and haematopoietic system system [17]. Later [17]. inLater the in same the year,same theseyear, these publications publica- weretions followed were followed by a report by a report showing showing that RAF1 that RAF1 counteracts counteracts apoptosis apoptosis by binding by binding to and to inhibitingand inhibiting the proapoptotic the proapoptotic kinase kinase ASK1 ASK1 without without the need the forneed RAF1 for RAF1 catalytic catalytic activity activity [18]. Hüser[18]. Hüser et al.also et al. showed also showed that knocking that knocking out RAF1 outincreased RAF1 increased apoptosis apoptosis in embryonal in embryonal tissues withouttissues without affecting affecting ERK activation, ERK activation, and that and expression that expression of a RAF1 of mutanta RAF1 whichmutant cannot which be activated could rescue the apoptosis defect [16]. These results strongly suggested that Genes 2021, 12, 553 3 of 16 RAF1 counteracts apoptosis
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