mechanisms of ret signaling in cancer: current and future implications for targeted therapy

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Cellular Signalling xxx (2014) xxx–xxx

CLS-08151; No of Pages 10

Contents lists available at ScienceDirect

Cellular Signalling

j ourna l homepage: www.e lsev ie r .com/ locate /ce l l s ig

Review

Mechanisms of RET signaling in cancer: Current and future implicationsfor targeted therapy

OFI. Plaza-Menacho a,⁎, L. Mologni b, N.Q. McDonald a

a Structural Biology Laboratory, London Research Institute, Cancer Research UK, London, UKb Dept. of Health Sciences, University of Milano—Bicocca, Italy

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⁎ Corresponding author at: Structural Biology LaboratCancer Research UK, 44 Lincoln's Inn Fields, London WC3360; fax: +44 20 7269 3259.

E-mail address: [email protected] (I.

http://dx.doi.org/10.1016/j.cellsig.2014.03.0320898-6568/© 2014 Published by Elsevier Inc.

Please cite this article as: I. Plaza-Menacho, e

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Article history:Received 8 March 2014Accepted 30 March 2014Available online xxxx

Keywords:RTKSignalingCancerKinaseSmall molecule inhibitors

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RDe-regulation of RET signaling by oncogenic mutation, gene rearrangement, overexpression or transcriptionalup-regulation is implicated in several human cancers of neuroendocrine and epithelial origin (thyroid, breast,lung). Understanding how RET signaling mechanisms associated with these oncogenic events are deregulated,and their impact in the biological processes driving tumor formation and progression, as well as response totreatment, will be crucial to find and develop better targeted therapeutic strategies. In this reviewwemake spe-cial emphasis on the distinct mechanisms of RET signaling in cancer and in the current knowledge related tosmall molecule inhibitors targeting the tyrosine kinase domain of RET as therapeutic drugs in RET-positivecancers.

© 2014 Published by Elsevier Inc.

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1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. The RET receptor system: implications for early development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03. RET wild-type mechanism of signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04. RET signaling in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4.1. RET signaling in multiple endocrine neoplasia type 2 (MEN2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.2. RET signaling in breast cancer: direct implications for endocrine therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.3. Signaling by oncogenic RET kinase fusions in Papillary Thyroid Carcinoma and in lung cancer . . . . . . . . . . . . . . . . . . . . . . . . 0

5. Targeted therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.1. Current RET inhibitors in clinical practice and research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.2. Future perspectives on RET kinase inhibition and clinical translation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

6. Conclusion and final remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

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UN1. Introduction

The RET receptor tyrosine kinase (RTK) is encoded by the RET proto-oncogene localized on human chromosome 10q11.2. It is the only RTKwith cadherin-like domains in its extracellular region. The fourcadherin-like domains contain a calcium binding site and precede acysteine-rich motif, a single membrane-spanning region, and an

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intracellular region containing an active tyrosine kinase domain withtwo flanking regulatory regions, the juxtamembrane and a C-tail(Fig. 1).

RET (Fig. 1) is the signaling receptor for the glial cell-derived neuro-trophic factor (GDNF) family of ligands (GFLs): GDNF, Neurturin,Persephin and Artemin [1]. These ligands play a key role in promotingsurvival, differentiation and chemotaxis of neurons [2,3] as well assome neoplastic epithelial cells [4–7]. In order to stimulate RET tyrosinekinase activity from the extracellular compartment, these GFLs firstneed to form a complex with their glycosylphosphatidylinositol (GPI)-anchored co-receptors, the GDNF receptor-α family (GFRα1–4), afterwhich the GFL–GFRα complex recruits and activates RET [2]. Different

), http://dx.doi.org/10.1016/j.cellsig.2014.03.032

http://dx.doi.org/10.1016/j.cellsig.2014.03.032

mailto:[email protected]


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Fig. 1. Diagram representation of the RET receptor tyrosine kinase with discrete functional domains: Cadherin like-domains (CLDs) 1 to 4, calcium binding site, cysteine-rich domain(CRD), single transmembrane domain (TM), intracellular tyrosine kinase domain (TK), juxtamembrane (JM) region and C-terminal tail (CT). Together with family of ligands: GDNF,ARTN, NRTN, PSPN and GPI-anchor co-receptor GFRα [1–4]. Solid arrows indicate preferred binding. Please note that some degree of crosstalk has been shown, see ref [2].

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models of receptor assembly and activation have been proposed de-pending on the pattern of receptor and co-receptor colocalization [1].The GFRαs exhibit preferences for specific GFLs, although there is tosome extent crosstalk among them (Fig. 1).

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Based on genotype–phenotype correlations observed in differentinherited human diseases and syndromes [8], and in transgenic mousemodels of disease where the different components of the RET receptorsystemhave been geneticallymanipulated, it is established that RET sig-naling is crucial for kidney organogenesis and the formation of neuralcrest derived lineages, in particular neuronal populations of the periph-eral and central nervous system.

In knockout studies Ret inactivation resulted in renal agenesis due tofailure of the ureteric bud to invaginate properly from theWolffian duct[9,10]. Knock-in mice studies have provided evidence for differentialisoform-specific roles of Ret in kidney development. Comparison be-tween mice expressing homozygote Ret9 (Ret9+/+) and mice express-ing Ret51+/+ showed severe kidney defects by the latter, whereasRet9+/+ mice were normal [11]. In contrast, a later study reported thatmice homozygous for either Ret9 or Ret51were viable and showed nor-mal kidneys [12]. Interestingly, mutation of Tyr1015 impacted on theureteric bud outgrowth in the context of either isoform, providing evi-dence for the importance of PLCγ signaling downstream from Ret inrenal development.

Hirschsprung's disease is a genetic disorder affecting neural crest de-velopment characterized by the absence of enteric parasympatheticneurons along the intestinal tract. Ret is expressed in several neuronalsubpopulations derived from this stem cell lineage, including cells inthe enteric, sensory, and sympathetic nervous systems [13]. Homozy-gote mice for a targeted mutation disrupting the Ret kinase domain(RetKD+/+) lack enteric neurons throughout the intestinal tract [9]. Re-cently, two conditional Ret and the Gfrα1 targeted knockout inpostmigratory enteric neurons were reported to cause neuronal death

Please cite this article as: I. Plaza-Menacho, et al., Cellular Signalling (2014

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leading to aganglionosis of the intestinal track, a phenotype mimickingHirschsprung's disease [14,15].

Ret is also expressed in motoneurons from the spinal cord whereGdnf has potent pro-survival effects in the earliest stages of embryonicdevelopment [13,16] and also during late embryogenesis [17]. Duringearly development, Ret is also required for the correct projection ofhind limb-innervating axons in a process that requires the cooperationwith EphA4 [18]. In addition, Gdnf acts as a pro-survival factor in ventralmidbrain dopaminergic neurons [19]. Ret is expressed at high levels inadult ventral midbrain dopaminergic neurons of the substantia nigra,and exogenous application of Gdnf was shown to protect Ret-expressing neurons in an animal model of Parkinson's disease [20].

Previous studies also evaluated the genetic manipulation of GDNFfamily of ligands and co-receptors in mouse models. The first describedGdnf knockoutmice demonstrated abnormalities in both peripheral andcentral noradrenergic neurons, whereas the mesencephalic dopamineneurons remained intact [21–23]. Whereas the Gfrα1 knockout miceshowed that Gfrα1 is required for the development of distinct subpop-ulations of motoneuron [24] aswell as to promote (together with Gdnf)the differentiation and tangential migration of cortical GABAergic neu-rons [25]. Interestingly Gdnf and Gfrα1 are not essential for the devel-opment of satellite glia in mouse sympathetic ganglia [26], Howeverin a recent study, a critical role of Gfrα1 in the development and func-tion of themain olfactory systemwas uncovered [27], which contrastedwith the alimentary tract innervation deficits and dysfunction observedin mice lacking Gfrα2 [28]. Another study reported that Nrtn signalingvia Gfrα2 is essential for innervation of glandular but notmuscle targetsof sacral parasympathetic ganglion neurons [29]. Nrtn-deficient micedeveloped dry eye and keratoconjunctivitis sicca [30]. The characteriza-tion of Artn- and Gfrα3-deficientmice revealed similar abnormalities inthe migration and axonal projection pattern of the entire sympatheticnervous system. Artn is expressed along blood vessels and in cells near-by to sympathetic axonal projections. In the developing vasculature,Artn is expressed in smooth muscle cells of the vessels, and it acts as aguidance factor that encourages sympathetic fibers to follow blood ves-sels as they project toward their final target tissues. Mice lacking Pspn



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showed normal development and behavior, but are hypersensitive tocerebral ischemia [31]. Gfrα4-deficient knockout mice showed no dif-ferences in growth, gross behavior, or viability. The number and mor-phology of the thyroid C cells were indistinguishable between thegenotypes in both newborn and adult age. However, thyroid tissue cal-citonin content was reduced by 60% in new-borns and by 45% in 3 weekold Gfrα4-knockout mice. In contrast, thyroid calcitonin levels weresimilar in adult animals. Consistent with the reduced calcitonin levels,bone formation rate in juvenile Gfrα4 knockout mice was increased.This study indicates a novel role for endogenous Gfrα4 signaling in reg-ulating calcitonin production in thyroid C cells of young mice [32].

In this review special emphasis is made on the distinct mechanismsof RET signaling in cancer, not only those promoted by oncogenicderegulation caused by point mutations and chromosomal transloca-tions (i.e. oncogenic RET fusions), but also thosemechanisms associatedwith up-regulations of the wild-type receptor (i.e. breast cancer).

3. RET wild-type mechanism of signaling

Upon ligand and co-receptor binding, RET is recruited and assem-bled into a multicomponent receptor complex, followed by activationof the kinase domain and autophosphorylation of intracellular tyrosineresidues. These phospho-tyrosine residues then serve as docking sitesfor downstream signaling proteins carrying SRC homology 2 (SH2) orphosphotyrosine-binding (PTB) domains mainly, which transmit sig-nals into the cell via the activation of a wide range of effector proteins[8].While the latter role for autophosphorylation has beenwidely dem-onstrated for RET, the effect of autophosphorylation on catalytic activa-tion is less clear. At least 14 of the 18 tyrosine residues present in theintracellular region of RET can become phosphorylated [33–35]. Invitro, activation loop phosphorylation has little effect on catalytic activ-ity despite the fact that they are known to contribute to full kinase acti-vation in cells [34]. These residues have been shown to be redundant forRET autophosphorylation in vitro usingpurified recombinant intracellu-lar domain of RET [35,36], despite evidence that they are crucial for RETsignaling in cells [34]. These data indicate that the cellular effects linkedto individual tyrosine autophosphorylation sites are not a direct conse-quence of the lack of kinase activity by the receptor per se, but due to thedisruption of downstream signaling pathways and feedback loop at theintracellular level. Autophosphorylation of Tyr1062 is required for acti-vation of the RAS/ERK1/2 and PI3K/AKT pathways [37–40]. This residueis critical for RET function in cells and also in vivo, as mice with a pointmutation in Tyr1062 to phenylalanine show a severe loss-of-functionphenotype [12,41]. Upon ligand binding and receptor activation, atleast two distinct sets of protein complexes can assemble on phosphor-ylated Tyr1062 via SHC. One activating the RAS/ERK1/2 kinasepathway through recruitment of GRB2 and SOS, and another activatingthe PI3K/AKT pathway through recruitment of adaptors GRB2 andGAB2followed by p85PI3K and the SHP2 tyrosine phosphatase [37]. The adap-tor protein FRS2 can also bind to phosphorylated RET Tyr1062 [42,43].FRS2 competes with SHC for binding to Tyr1062, and this may bemedi-ated from different compartments in the plasma membrane, as RET isknown to interact with FRS2 in lipid rafts, and with SHC outside lipidrafts [44]. Adaptor molecules from the p62DOK family have also beenshown to interact with RET. Among them, DOK [1–6], interactswith phosphorylated Tyr1062 via their PTB domains [45,46]. TheGRB2/GAB2 complex can also directly interact with phosphorylatedTyr1096 (present only in RET51), as an alternative route to PI3K activa-tion by GDNF. Autophosphorylation of Tyr1015 leads to activation ofPLCγ [47], and Tyr981 results in Src non-receptor tyrosine kinase bind-ing [48]. Recently, the GTPase activating protein (GAP) for Rap1,Rap1GAP was identified as a novel RET-binding protein. Rap1GAP re-quires phosphorylation of Tyr981 for RET binding and suppressedGDNF-induced activation of ERK1/2 and neurite outgrowth [49]. In thejuxtamembrane region, RET Y687 is the binding site for SHP2 [50].This interaction also required RET Y1062 downstream signaling


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components for the activation of the PI3K/AKT pathway and to promotesurvival and neurite outgrowth in primary neurons. In addition, phos-phorylation at RET S696 by protein kinase A (PKA) has also been report-ed. Mutation of S696A affected the ability of RET to activate the smallGTPase Rac1 and stimulate formation of cell lamellipodia [51]. Homozy-gous knock-inmice carrying thismutation lacked enteric neurons in thedistal colon, resulting from a migration defect of enteric neural crestcells [52], indicating a physiological role for a PKA–RET functionalcrosstalk. These findings established RET autophosphorylation siteY687 as an important autophosphorylation site for the integration ofRET and PKA signals.

One interesting aspect of RET signaling is the direct interaction ofwild-type RET with downstream signaling partners independent of theautophosphorylation process. Among them, SHANK3, a PDZ domain-containing protein was found to interact with a PDZ-binding motifpresent in the RET9 sequence (FTRF) but not in the RET51 isoform. Thisinteraction was required to mediate sustained ERK1/2 and PI3K signal-ing, that resulted in enhanced branched tubular structures in 3D-cultures of epithelial cells [53]. In addition a recent study revealed thatthe FERM-domain of FAK binds to the unphosphorylated RET kinase do-main priming a direct and reciprocal RET–FAK phosphorylation mecha-nism in trans [54]. In particular, RET kinase was able to increase FAKactivation loop Y576/577 phosphorylation levels, and FAK was able inturn to increase phosphorylation levels of RET on activation loop Y905.Crucially FAK was able to rescue the RET kinase impaired mutantK758M [54]. This reciprocal and direct phosphorylation was observedalso in medullary thyroid carcinoma cells, indicating that FAK inhibitorscould be used as potential therapeutic agents for patients with multipleendocrine neoplasia type 2 tumors because both, treatment with theFAK kinase inhibitor NVP-TAE226 and FAKdown-regulation by siRNA re-duced RET phosphorylation and signaling aswell as the proliferation andsurvival of tumor and transfected cell lines expressing oncogenic RET[54]. In the same line, the apparent defect in intrinsic kinase activity forRET Y981F, Y952F, or Y928F mutants observed in transfected cells, butnot the reduction in activity of RET Y905F, could be partially repairedor restored by c-SRC or, more extensively, by v-SRC, which promotedY905 phosphorylation in trans. A complex was shown to be formed be-tween v-SRC and RET in the lipid rafts. Finally, endogenous c-SRC wasshown to promote Y905 phosphorylation of RET Y981F mutant in vivoindicating a novel SRC kinase-mediated repair mechanism of function-impaired mutant RET kinases [55]. These studies emphasize the impor-tance ofmechanisms that regulate RET kinase function from the intracel-lular compartment (independent of ligand stimulation), an area that hasbeenunderstudied, and to the same extent underestimated. In the case ofRET this may lead to the identification of novel RET kinase regulatorymechanisms in trans by novel RET signaling complexes.

4. RET signaling in cancer

4.1. RET signaling in multiple endocrine neoplasia type 2 (MEN2)

Despite the clear genotype–phenotype correlation found in MEN2,the molecular mechanisms that connect the specific mutant receptorswith their different clinical subtypes (MEN2A, MEN2B and FamilialMedullary Thyroid Cancer, FMTC) are far from understood while muta-tions affecting the extracellular cysteine-rich domain of RET (associatedwith MEN2A and FMTC) result in covalent dimerization and constitu-tively activation of the receptor [8]. Mutations affecting the intracellulardomain of RET, usually associated with FMTC and always with theMEN2B, signal independently of GDNF as monomeric oncoproteins.These oncoproteins show not only an altered catalytic activity but alsoan altered substrate specificity because, contrary to wild-type RET,they tend to phosphorylate substrates that are usually preferred by cy-toplasmic tyrosine kinases such us SRC, FAK and ABL [56]. One rationaleabout themolecular basis of this disease is that a different pattern of re-ceptor autophosphorylation by specific mutants connects a different set



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of phosphotyrosine-mediated downstream signaling pathways andtranscriptional programs associated with specific clinical phenotypes.To make the picture even more complex, there is evidence aboutGDNF further enhancing oncogenic RET activity with MEN2A andMEN2B mutations [57], and paradoxically that gain of function (onco-genic) mutations could also be associated with Hirschsprung's disease(HSCR), a developmental disorder linked with RET haploinsufficiency[8,57]. In an attempt to resolve this apparent paradox, several groupsproposed that a critical threshold level of RET activity is necessary topromote neural crest survival. In this model, RET C609Y and RETC620R mutants display a tyrosine kinase activity under the thresholdrequired to promote neuroectodermic cell survival [58]. Alternatively,it has been assumed that these twomutants trigger a signaling pathwaythat commits the cell to death [59,60]. Howeverwhen these RET geneticvariants were expressed in SK-N-MC cells, they exhibited a constitutivetyrosine kinase activity higher than the wild-type protein butcontrary to the main MEN2A mutation (i.e. C634R) they were weaklytransforming and did not by themselves caused cell death. Furthermore,addition of GDNF did not increase RET C609Y and RET C620R phosphor-ylation levels or induce the activation of PI3K/AKT and RAS/ERK1/2.Consistently, GDNF could not protect the SK-N-MC cells expressing nei-ther RET C609Y nor C629R from anisomycin-induced apoptosis. Crucial-ly this was correlated with the intracellular accumulation of thesemutants under a 150-kDa incompletely glycosylated form and the lackof GDNF responsiveness.

Despite an incomplete picture for the autophosphorylation patterndisplayed by wild-type and oncogenic RET receptors, the notion that adifferent pattern of receptor autophosphorylation connects a differentset of phosphotyrosine-mediated downstream signaling pathways totranscriptional programs associated with specific clinical phenotypeshas been supported by several studies over the past few years. In cell-based experiments RET M918T (MEN2B) oncoproteins trigger elevatedlevels of PI3K activity and its downstream signaling cascade [61]. In ad-dition, expression of RET/PTC2 with a MEN2B mutation, compared towild-type RET/PTC2, switches the specificity of the RET tyrosine kinasetoward Crk and Nck cellular substrates that resulted in activation of thecytoskeleton protein Paxillin [62] and elevated levels of Jun N-terminalkinase activity JNK [63]. Along the same lines, RET M918T (MEN2B)showed enhanced phosphorylation of RET Tyr1062 and its associationwith SHC compared with RET C634R (MEN2A) mutants, resulting inhigher activation of the RAS/ERK1/2 and the PI3K/AKT signaling path-ways [64]. Transactivation of STAT3 by RET C634R (MEN2A) is requiredfor cellular transformation in a process that is independent of non-receptor tyrosine kinases JAKs and SRC [65]. By contrast, activatingpoint mutations targeting the kinase domain RET Y791F and RETS891A (FMTC) implicate JAKs and SRC kinases in the constitutive activa-tion of STAT3 [66]. Moreover, Yuan et al. [67] showed that oncogenicRETM918T (MEN2B) interactedwith and activated STAT3more strong-ly than RET C634R (MEN2A). These data support previous findingsshowing that RET MEN2B mutants do preferentially associate withSRC, of which STAT3 is one of the best-characterized targets [68]. Inthe same line, oncogenic RET enhanced in cells the phosphorylationlevels of FAK activation loop Tyr576/577 phosphorylation comparedto wild-type RET. In the case of oncogenic mutations targeting the ki-nase domain of RET (i.e. RET M918T, MEN2B), this phosphorylationevent was more robust than those mutants targeting the extracellulardomain (i.e. RET C634R, MEN2A). Interestingly, this pattern was mir-rored by levels of SRC kinase activation promoted by RET, which inthe case of mutation affecting the extracellular domain (i.e. C634R)were very low whereas RET mutations targeting the kinase domainand associated with the MEN2B phenotype displayed robust levels ofSRC Tyr416 phosphorylation indicating a close relationship betweenlevels of RET, FAK, SRC and STAT3 activation. Another interesting obser-vation is that oncogenic RET seems to be heavily internalized, and treat-ment with specific tyrosine kinase inhibitors (i.e. sorafenib) induced alysosomal degradation independent of proteosomal targeting [69].


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Furthermore, there is evidence that oncogenic RET M918T (MEN2B)signals in the endoplasmic reticulum and that the immature non-glycosylated form (i.e. precursor) of RET M918T (MEN2B) becomesphosphorylated already during synthesis in the endoplasmic reticulum[70]. At least three different tyrosine residues (Tyr905, Tyr1062,Tyr1096) of the RET M918T precursor are phosphorylated before theoncogenic receptor reaches the cell surface. Furthermore the authorsalso demonstrate that the precursor of RET M918T interacts with bothgrowth factor receptor and SH2-domain containing proteins alreadyin the endoplasmic reticulum, and that this interaction is dependenton the kinase activity of RET. Taken together, these studies show thecomplexity of RET (oncogenic) signaling, in a way that localization ofthe receptor in the cellular compartments as well as the interactionwith the extracellular environment may be important elements to un-derstand the function of RET, and howoncogenicmutations can perturbit in many different ways.

Despite the intensive study on RET signaling and cell biology overthe last two and a half decades, the molecular basis for RET receptor ty-rosine kinase activation and oncogenic deregulation has remainedlargely unclear. RET belongs to a small group of receptor tyrosine ki-nases whose kinase domain conformation and catalytic activity do notdepend on activation loop phosphorylation, similar to the EGFR andnon-receptor tyrosine kinase ACK1 [71,72]. Recent progress on thestructural basis for EGFR activation has emphasized the importance ofprivatemechanisms of activation for receptor tyrosine kinases involvingkinase domain flanking regions and the formation of oligomeric ar-rangements of their kinase domains. The activation mechanism operat-ing in RET has not been properly investigated in those terms. Similarly,only limited studies have examined how oncogenicmutation of the RETkinase domain leads to deregulation of kinase domain activity (usingmainly transfected cell lines or the only two available primary tumorcell lines as experimental system). Previous studies have focused eitheron the higher activity of oncogenic RET mutations or changes to RETsubstrate specificity [54,66,67]. In a recent study by Plaza-Menachoet al. [36] insights into the molecular mechanisms of RET kinase activa-tion and oncogenic deregulation have been elaborated. In this study, thetemporal pattern (kinetics) of RET autophosphorylation was defined bylabel-free quantitative mass spectrometry (LFQMS). The authors foundthat upon kinase domain activation the earliest detectable autophos-phorylation sites map to regions flanking the kinase domain core,while sites within the activation loop of the kinase only form at latertime-points. Comparison with oncogenic RET kinase revealed that lateautophosphorylation sites (i.e. those located at the activation loop) be-comephosphorylatedmuch earlier thanwild typeRET,which is due to acombination of enhanced enzymatic activity, increased ATP affinity, andsurprisingly to enhanced RET kinase substrate presentation in trans. Inthis study structures of oncogenic M918T and wild type RET kinase do-mains were presented revealing two discrete glycine-loop conforma-tions, one is an “open” ATP-competent, while the second “closed”conformer defined an unknown cis-inhibitory mechanism by stericallyblocking ATP-binding via a tether of hydrogen bonds between E734from the glycine rich loop and R912 from the activation loop, whichalso interacted with D771 from the αC helix. Mutations affecting thiskey glycine-loop tether perturb the autophosphorylation trajectory,but contrary to oncogenic mutations, they uniquely enhance RET sub-strate presentation in trans. The unexpected finding that oncogenicRET not only increases RET enzymatic activity but also generates a bet-ter substrate in trans that is less stable but binds ATP with higher affin-ity, illustrates the subtlety and complexity of themolecularmechanismsgoverning RET kinase activation and oncogenic deregulation.

4.2. RET signaling in breast cancer: direct implications for endocrine therapy

Over the last decade there has been considerable interest insearching for novel therapeutic targets in breast cancers, not only andspecifically oncogenic drivers of the disease but also modulators of



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response to therapy. Receptor tyrosine kinases have emerged as an im-portant family of proteins implicated in the modulation of response totherapy. Despite few tyrosine kinase-activating mutations having beenidentified in breast cancers, many breast cancers are characterized byamplification or overexpression of receptor tyrosine kinases such asHER2, EGFR, IGFR and FGFR1 that can drive tumor growth [73]. Two in-dependent studies recently identified RET overexpression in a subset ofERα-positive breast cancers [4,74], and further studies in the same co-hort of patient samples corroborated that increased RET mRNA levelscorrelated with increased RET protein expression [5]. These findingshighlight the potential implication of RET activation and signaling inthe etiology and development of ERα-positive breast tumors. Crucially,RET is involved in the biology of breast cancers in mouse models[75–77]. In GDNF-treated MCF7 cells (an ERα/GFRα1/RET-positivebreast cancer model) estrogen independent RET signaling results in in-creased ERα phosphorylation at Ser118 and Ser167 and transcriptionalactivation of ERα-dependent genes protecting breast cancer cells fromthe antiproliferative effects of tamoxifen, a drug targeting the estrogenreceptor [5]. RET mediated ERα phosphorylation occurs predominantlyvia themTOR/p70S6K pathway as it is blocked by rapamycin treatment.Whereas chemical inhibition of the AKT and ERK1/2 pathways had littleimpact on ERα phosphorylation, a small but reproducible inhibition ofERα Ser118, but not Ser167 phosphorylation, was observed followingPI3K inhibition. These data indicate that mTOR activation downstreamof RET signaling is not exclusively mediated via AKT. Certainly themechanism bywhich RET signaling results inmTOR activation indepen-dently of AKT is worthy of further investigation, because targeting RETdownstreamsignaling pathwaysmight be a therapeutic option in breastcancer treatment. Importantly, the crosstalk between the RET and ERαis important as a signaling axis promoting resistance to tamoxifen inMCF7 cells. RET siRNA silencing enhances the response of parental (re-sponsive) MCF7 cells to the drug and restores a tamoxifen-sensitivephenotype in tamoxifen-resistant variants [5]. In parallel to the involve-ment of GDNF/RET axis in tamoxifen resistance, ectopic expression ofanother RET ligand ARTN in MCF7 cells induces resistance to anti-estrogenic agents (tamoxifen and fulvestrant) in vitro and to tamoxifenin xenograft models [78]. These results are interesting, because ARTNexpression is estrogen inducible and correlates with reducedmetastasis-free and overall survival in tamoxifen treated ER-positivebreast cancer patients [78]. However, whether targeting RET in thesemodels blocks ARTN-induced tamoxifen resistance and whether GDNFand ARTN promote alternate downstream RET signaling have to be fur-ther investigated. A recent study by Spanheimer and co-workers dem-onstrate that knockdown of TFAP2C or RET inhibited GDNF-mediatedactivation of ERK1/2 and AKT in MCF-7 cells. In addition, targeting RETby both, siRNA or sunitinib, blocked GDNF-mediated activation ofERK1/2 and AKT in vitro and in vivo using a xenograft model. Inhibitionof RET by treatment with tyrosine kinase inhibitors sunitinib or vandet-anib reduced RET-dependent growth of luminal breast cancer cells. In-terestingly, knockdown of TFAP2C, which controls at transcriptionallevel both ERα and RET, demonstrated a greater effect on cell growththan either RET or ERα alone. Experiments using treatmentwith tamox-ifen and sunitinib confirmed the increased effectiveness of dual inhibi-tion of the ERα and RET pathways in regulating cell growth. Whereastargeting the ERα pathway altered cell proliferation, RET targeting pri-marily increased apoptosis in xenografts [79]. These findings highlighta therapeutic window for combinatorial therapy with antiestrogenand compounds targeting RET in luminal breast cancer. Furthermore,in models of breast cancer cells 2D and 3D culture, GDNF-mediatedRET signaling is enhanced in a model of Aromatase Inhibitor (AI) resis-tance [7]. Further, GDNF-RET signaling promoted the survival of AI-resistant cells and elicited resistance in AI-sensitive cells. Both theseeffects were selectively reverted by the RET kinase inhibitor NVP-BBT594. Gene expression profiling in ERα-positive cancers defined aproliferation-independent GDNF-response signature associated with apoor patient outcome and, more importantly, predicted poor response


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to AI treatment with the development of resistance. We validatedthese findings by demonstrating increased RET protein expressionlevels in an independent cohort of AI-resistant patient specimens. To-gether, these data establish the GDNF-RET signaling as a rational thera-peutic target to combat or delay the onset of AI resistance in breastcancer. These recent studies are the bases for building a consensus inthe breast cancer field pointing at RET as a new therapeutic target inbreast cancers, a fact that has been further supported by the study ofGatelli et al. [80] were they show that ligand-induced Ret activationstimulates both migration of breast cancer cells and expression of cyto-kines in the presence of endocrine agents. A positive feed-forward loopbetween the inflammatory cytokine IL-6 and Ret was required forin vivo metastasis potential and mediated by FAK.

4.3. Signaling by oncogenic RET kinase fusions in Papillary Thyroid Carcinomaand in lung cancer

The clinical relevance of RET in human diseases was first recognizedin Papillary Thyroid Carcinoma (PTC), the most prevalent thyroid can-cer, accounting for 80 to 90% of all thyroid malignancies [81]. Thereare several somatic genetic lesions associated with PTC, including chro-mosomal alterations that affectNTRK1 and RET, togetherwith oncogenicactivation of the RAS, BRAF,MET, TSH-R,GSA, and p53 genes [81]. Specificrearranged forms of RETwere detected in PTC [82] giving rise to fusiononcogenes encoding proteins that harbor the intracellular kinase do-main of RET and the N-terminal domain of various proteins. The N-terminal domains of these various unrelated proteins all have, in theory,the potential property to enhance dimerization of the resulting fusionprotein, leading to autophosphorylation of tyrosine residues in the tyro-sine kinase domain of RET. Almost exclusively, the breakpoints in REToccur at sites distributed across intron 11 [83], giving rise to proteinswithout a transmembrane domain and juxtamembrane region. Theseoncogenic fusions encode constitutively active cytoplasmic chimericproteins referred to as RET/PTCs. To date, more than 12 different fusionpartner genes, and due to variable breakpoints, at least 17 different REThybrid oncogenes are reported [84,85]. The most prevalent variants ofthese chimeric oncogenes are RET/PTC1 (i.e. CCDC6–RET oncogenic fu-sion, 60 to 70%) and RET/PTC3 (i.e. NCOA4-RET oncogenic fusion, 20 to30%) [86,87]. The etiology and molecular mechanisms driving PTC arenot fully understood. However it is clear that several key genetic eventshave been implicated in thyroid tumorigenesis. Point mutations in theBRAF or RAS genes or rearranged in transformation RET/PTC gene rear-rangements are observed in approximately 70% of papillary cancercases. Consequently, deregulation of the ERK1/2 pathway is a mainaxis driving the oncogenic phenotype [88] in addition to the PI3K/AKTpathway, which is overactivated preferentially by RET-PTCs (i.e. RET-PTC1 and 3) via the phosphorylation and recruitment of novel thyroidspecific adaptor proteins, e.g. XB130 [89]. In the case of RET-PTC3, en-hanced basal and insulin-stimulated PI3K/AKT signaling increases IRS2expression in thyroid cells [90]. A strong inflammatory response pro-moted by RET-PTCs via STAT1 activation has also been observed [91].Moreover, NFkB has been recently shown to play an important role inthyroid cancer for its ability to control the proliferative and the anti-apoptotic signaling pathways of thyroid neoplastic cells [92,93] in addi-tion to the finding that the beta-catenin/Tcf4 axis appears to bederegulated by these oncogenic RET kinase fusions [94,95]. For furtherdetails on the different signaling pathways activated in follicular cell-derived thyroid carcinomas by PTC oncogenic fusions see [96].

The recent identification of oncogenic KIF5B–RET, CCDC6–RET andTRIM33–RET fusions in lung adenocarcinoma has directed attention toRET as a new therapeutic target in lung cancer [97,98]. In an unselectedpopulation of NSCLCs, RET fusions are present in 1–2% of cases. Howev-er, this incidence increases substantially to 16 to 17% in never-smokercases lacking other known oncogenic alteration, defining an exclusivemolecular subtype [99–103]. Although in vitro and xenograft modeldata provided experimental support for the potential of RET inhibitors



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in the treatment of RET fusion-positive tumors, information about mo-lecular and biological mechanisms by which these oncogenic RET chi-meras exert their transforming action is lacking. The understanding ofsuch mechanisms is crucial to find optimal therapeutic options, as cur-rent compounds targeting RET kinase that are tested in the clinic arenot fully specific anddo not provide a substantial overall survival advan-tage [104].

5. Targeted therapies

The advent of next generation sequencing has started a revolution inthe molecular classification of cancer. Tumors are now beginning to becatalogued as genetic rather than histologic entities. Accordingly, cancerpatients are being stratified per molecular defect rather than tumor his-tology. The great success of targeted therapies has fostered this changeof view. Along these lines, RET is no longer solely the ‘thyroid canceroncogene’, since it has been described as a driver oncogene in lung can-cers as well as an important molecular determinant in other cancers ofepithelial origin (i.e. ERα positive breast cancer). An updated descrip-tion of clinical results obtained in clinical trials involving RET-positivecancer patients is given in the following paragraph. For preclinicaldata, the reader is referred to a recent review [105]. In this review wespecifically focus on small molecule inhibitors targeting the tyrosine ki-nase domain of RET.

5.1. Current RET inhibitors in clinical practice and research

Protein kinases represent the largest family of druggable targets inoncology. Some of the most recent advances in cancer therapy arebased on the use of small compounds targeting the nucleotide-bindingpocket of a kinase to block phospho-transfer activity. Imatinib was thefirst clinically approved TKI in the early 2000s. As imatinib seemed tohave some anti-RET activity [106], it was tested in MTC patients [107].However, the results were disappointing, due to poor activity of thedrug against RET kinase, compared to its primary targets ABL, PDGFRand KIT. Since then, a large effort in both basic and preclinical researchhas been spent to find (but crucially not to design, see next) selectiveRET kinase inhibitors [108]. Eventually, vandetanib (ZD6474,Caprelsa®), a quinazoline developed by AstraZeneca initially as aVEGFR inhibitor [109], was approved in 2011 for the treatment of locallyadvanced or metastatic MTC, after encouraging results from the ZETAtrial [110]. The drug is effective against most oncogenic RET mutants,but not the gatekeepermutant V804L/M [111,112]. The crystal structureof the RET kinase domain bound to vandetanib has been determined[35] and showed that the bromo-fluoro-phenyl moiety of the com-pound inserts deep into the selectivity pocket adjacent to the gatekeep-er residue, explaining why substitution with a bulky amino acid causesresistance (Fig. 2). On the other hand, the bromine atom establishes vander Waals contacts with the back of the cavity, thus stabilizing kinase–drug interaction and providing selectivity (Fig. 2). Vandetanib bindsunphosphorylated and phosphorylated RET with equal affinity,explaining its inhibitory activity against hyper-active mutants, such asRET M918T (MEN2B) and C634W/R (MEN2A) RET. Clinical efficacywas demonstrated in 331 patients receiving vandetanib (300 mg/day)or placebo (ClinicalTrials.gov NCT00322452, ZETA trial). Progression-free survival (PFS) was significantly increased (19.3 months vs not-reached [estimated 30.5 months] in placebo and vandetanib groups, re-spectively; hazard ratio [HR], 0.46). Overall response rate (ORR) wasalso higher in the vandetanib arm (45%) compared to placebo (13%)as well as disease control rate (defined as ORR + stable disease; 87%vs 71%). Interestingly, breakdown of response data showed that RETmutation-positive patients had a better outcome compared to RETmutation-negative or -unknown patients, thus confirming that efficacywas specifically related to RET inhibition. This is important because van-detanib activity against VEGFR and EGFR kinases may confound inter-pretation of the data. Diarrhea, rash, nausea and hypertension were


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the most common side effects, leading to discontinuation in 12% of thepatients [104].

In 2012, a second RET inhibitor was approved forMedullary ThyroidCarcinoma (MTC). Cabozantinib (XL-184, Cometriq®) from Exelixis is amultikinase inhibitorwith a quinoline core [113,114]. Itwas initially de-scribed as a dual VEGFR/MET inhibitor with potent anti-angiogenicproperties. However, it also targets other kinases with similar potency,including RET. Cabozantinib showed promising results in MTC patientsduring a phase I/II trial on various solid tumors [115]. Therefore, a spe-cific phase III registration trial was then designed for advanced MTC(EXAM trial, NCT00704730). A total of 330 patients were randomizedto treatment (140 mg/day) or placebo. Median PFS was 11.2 monthsin the treatment arm versus 4.0 months in the placebo arm; HR, 0.28.The secondary endpoint, ORR was 27% vs 0%. Of note, this patient pop-ulationwas selected to have progressedwithin the previous 14 months,to exclude indolent disease. Common adverse events included diarrheaand palmar–plantar erythrodysesthesia and accounted for discontinua-tion in 16% of cases in the cabozantinib group. Four deaths were attrib-uted to VEGFR inhibition, highlighting the importance of targetselectivity [116]. Recent data on three RET-positive lung cancer patientssuggested good clinical activity of cabozantinib in this setting [100]. Twopatients showed durable partial response (PR) to treatment, while thethird had a prolonged stabilization of the disease (SD). Interestingly,one of the patients carried a novel TRIM33–RET fusion. All threepatients were progression-free at the time of report (4 to 8 months,still on treatment).

Several other drugs have reached clinical evaluation on RET-positivethyroid cancer, although with varying results, as illustrated below.Amgen developed motesanib (AMG-706) for the treatment of solid tu-mors. As RET is among its targets, motesanib was tested in a phase IIstudy in MTC and in differentiated carcinoma, mainly PTC [117,118];trial no. NCT00121628. The results were not so impressive, with ORRof 14% (PTC/FTC) and 2% (MTC). Stable disease was achieved in 67%and 81% of the patients, respectively. It should be noted that in vitro ac-tivity of motesanib against RET is less potent compared to vandetaniband cabozantinib [119].

Sunitinib (Sutent®, SU11248) is one of the most successful com-pounds from a large series of indolinone inhibitors designed by Sugen,now part of Pfizer. Sunitinib has reached clinical approval for three dif-ferent indications: pancreatic, kidney and gastrointestinal stromal tu-mors [120]. In these cases, its broad activity against VEGFRs, PDGFRsand KIT is exploited. Moreover, Sutent is also a potent RET inhibitor[121]. It has been evaluated in a small phase II clinical trial with promis-ing results [122]. Out of 33 evaluable patients, the authors reported 1complete response (CR) and 10 PR (33% ORR), while 16 patients hadSD, with an overall disease control rate of 82% and a median PFS of12.8 months. These data encouraged further investigation. Additionaltrials are currently evaluating sunitinib in thyroid malignancy(see www.clinicaltrials.gov).

Sorafenib (Nexavar®, Bayer) is a urea-based RAF inhibitor with sig-nificant anti-RET activity [69,123,124]. Based on its profile, sorafenibwas tested in phase II trials in unselected thyroid cancer patients [125]and in RET-positive metastatic MTC, hereditary (arm A) or sporadic(arm B) [126]. In the first trial, 23% response rate was reported, butthe figure is confounded by anti-BRAF activity, as BRAF mutations arecommon events in differentiated thyroid cancer and the authors didnot mention any mutational status. The latter trial reported a 6% RRand 88% SD in arm B, with acceptable toxicity. Despite low responserate by RECIST criteria, all patients experienced some degree of tumorshrinkage. Indeed, median PFS was 17.9 months. Recently, data from alarge phase III trial were disclosed at the 2013 ASCO meeting [127].The trial enrolled 417 metastatic patients with radioactive iodine-refractory differentiated thyroid cancer and randomized them tosorafenib or placebo. PFS was 10.8 months (sorafenib) vs 5.8 months(placebo; HR 0.58). RR in the sorafenib vs placebo arms was 12.2% and0.5% (p b 0.0001). However, no RET mutation analysis is reported.


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Fig. 2.Molecular insights for type I and type 2 RET tyrosine kinase inhibitors. (A) Crystal structure of phosphorylated RET kinase domain bound to vandetanib (ZD6474) (PDB ID: 2IVU)displaying also a modeled V804Mmutation. Discrete color code for inhibitor (orange) and secondary structural components: hinge region (yellow), αC helix (purple), glycine-rich loop(GRL, pink) and activation loop (AL, green) is depicted. Close-up view (lower panel) shows some key residues in the active site of RET interacting with the inhibitor defined by the DFG-in(active) conformer. Note that the V804Mmutant would clash with vandetanib, explaining lack of activity by this drug against the gatekeeper residuemutant. (B) Crystal structure of ABLT315I kinase bound to ponatinib (AP24534) (PDB ID: 3IK3). Discrete color code as used in (A). Close-up view (lower panel) shows some key residues in the active site of ABL T315I kinaseinteracting with the inhibitor defined by the DFG-out (inactive) conformer. Note that compound AP24534 reaches deeper into the active site, which is more compacted than the RET ki-nase. (C) Close-up overviewof the active site of theABL T315I kinase-ponatinib structure (PDB ID: 3IK3, darker color) superimposed to the RET kinase-vandetanib structure (PDB ID: 2IVU,color code as in A and B), with a modeled gatekeeper V804M mutation (*). The gatekeeper and DFG residue side chains are shown by stick representation and discrete color code. Notethat: i) compound AP24534 is not compatible with the RET activation loop DFG-in conformer, ii) ZD6474 would not overcome the drug-resistant V804M mutation and iii) the largerAP24534 compound displays more flexibility, getting deeper into the active site and forcing the activation loop to adopt the DFG-out (inactive) conformation. The figure was generatedusing the PyMOL Molecular Graphics System.Adapted from [119].


Based on these results, the FDA has given priority review designation tosorafenib for the treatment of locally advanced ormetastatic radioactiveiodine-refractory differentiated thyroid cancer (DTC).

Ponatinib (Iclusig®, Ariad Pharmaceuticals) is a multi-target kinaseinhibitor that has been approved for drug-resistant BCR/ABL-positive leu-kemia [128]. Rational drug design guided its development against bulkygatekeeper mutants, thanks to a rigid carbon–carbon triple bond linker(see Fig. 2) [119,129]. Strong anti-RET activity (including V804M/L gate-keeper mutants) was recently characterized in vitro and in vivo[119,130]. Specific clinical trials for RET-positiveMTC andNSCLC patientshave been designed (NCT01838642 and NCT01813734). Molecular


confirmation of a RETmutation or translocation is required for eligibility.Final data are expected in 2016 and 2015, respectively.

Fostamatinib is a spleen tyrosine kinase inhibitor pro-drug devel-oped by AstraZeneca for autoimmune disorders. Strong RET inhibitionby Fostamatinib was later described, as an unwanted off-target effect.It has recently been evaluated in amulti-histology phase II study in var-ious solid tumors, including 5 thyroid cancer patients [131]. Since theresults were disappointing (no responses) the company dropped anyfurther development.

Overall multi-targeted kinase inhibitors (i.e. ATP-competitive) haverevolutionized the role of chemotherapy in cancer, and in the case of



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Medullary Thyroid Cancer, they have provided the first time tolerabletherapeutic option that can improve outcome in patients with progres-sive disease [116]. Drugs targeting RET kinase do also target othermembers of the RTK family, however clearly even such “dirty” kinase in-hibitors are clinically useful. In clinical trials, vandetanib improves PFScompared with placebo from 19.3 months to 30.5 months (ZETA trial)in patients with metastatic disease, whereas cabozantinib improvedPFS from 4.0 months to 11.2 months in populations with moreaggressive disease (EXAM trial) [116]. Despite encouraging results, afull remission remains elusive, adverse effects are frequent andthemolecular targets of those agents are unclear due to the lack of spec-ificity and the crosstalk. New approaches for the design of clinical trials(i.e. selection and patient stratification according to their genetic andmolecular profiles) and the development ofmore specific targeted com-pounds against RET tyrosine kinase are needed to find better and moreefficient therapeutic strategies.

5.2. Future perspectives on RET kinase inhibition and clinical translation

The majority if not all of RET kinase inhibitors tested in the benchand translated to the clinic so far have not been specifically designedto inhibit RET per se. These compounds were intended to target specifickinases but were found to collaterally hit RET in kinase screenings to as-sess compound potency and specificity. Most of them are ATP-competitive inhibitors (i.e. type I inhibitors), which recognize the activeconformation of the kinase or the so-called DFG-in conformer [132].This active conformation present one to three hydrogen bonds to theamino acids located in the hinge region of the target kinase, therebymimicking the hydrogen bonds that are normally formed by the ade-nine ring of ATP. Examples include among others vandetanib(ZD6474), PP1 and AZD0530 compounds [132]. On the other handtype II kinase inhibitors would recognize the inactive conformation ofthe kinase, or DFG-out conformer, shown to have a potent inhibitory ef-fect, i.e. sorafenib, ponatinib [69,119], despite initial poor anti-RET activ-ity seen for imatinib [106]. Interestingly, no crystal structures of RETkinase with type II inhibitors have been successfully solved so far, con-trary to the multiple crystal structures of RET kinase in complex withATP competitive inhibitors (i.e. type I or DFG-in) [35,133].

An effort in undertaking both, proper and carefully conducted drug-design and discovery projects focused specifically on RET kinase ratherthan finding inhibitors with off-target or collateral effects; and the pur-suit of newRET kinase crystal forms thatwill allow crystallization of com-plexes with type II inhibitors (i.e. DFG-out) will be key to obtain morepotent and specific RET kinase inhibitors. In this line, uncovering thestructural and molecular basis for RET kinase activation, cis-regulationand oncogenic deregulation will provide insights into complex mecha-nisms of regulation involving distant sites from the active pocket, but af-fecting kinase activity (i.e. allosteric sites). Recent studies point to thatdirection [36]. We shall be looking beyond the horizon then toward allo-steric inhibitors as a mean to design and develop even more potent andspecific RET kinase inhibitors, clinically successful, as these compoundsexhibit the highest degree of kinase selectivity because they exploit bind-ing sites and regulatory mechanisms that are unique to a particularkinase. In last place, but not less important, is the focus on the delineationof the signaling profiles associated with specific RET kinase domain mu-tants, whichmay provide an alternativemeans by using a combination ofdifferent compounds targeting, in addition to RET, important down-stream effectors driving the growth of the cancer cells.

6. Conclusion and final remarks

The discovery of the RET oncogene almost thirty years ago openedresearch avenues that havemade a profound impact in the fields of can-cer research, developmental biology, and neuroscience. RET is a remark-able and paradigmatic example illustrating howgeneticmutations, bothgain of function (oncogenic) found in thyroid cancer mainly and also in


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lung cancer; and loss of function mutations (inactivating) found inHirschsprung's disease, can impact in the function of the receptor hav-ing such profound biological and clinical consequences. In addition, re-cent studies have prompted that deregulation of the expression ofwild type RET is also an important molecular determinant in sometypes of cancers of epithelial origin (i.e. breast cancer). Less clear how-ever is the relevance of the GDNF family of ligands in this context, anddespite some recent advances further research is needed to elucidatethe role of the different RET ligands in the formation and progressionof RET-positive tumors. Uncovering the structural and molecular deter-minants of RET kinase activation and oncogenic deregulation, togetherwith an emphasis on proper drug-design and discovery project focuson RET and the pursuit of distinct crystal forms allowing novel RETkinase-inhibitor complexes (i.e. type II kinase inhibitors, or DFG-out),will be crucial to design and develop more specific and potent RET ki-nase inhibitors, which should be at the forefront of the new therapeuticstrategies for patients with RET-driven tumors, hopefully in the nearfuture.

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