therapeutic targeting of c-kit in...

1. Introduction

2. Mechanism of oncogenic KIT

activation

3. Signalling pathways

downstream of KIT

4. KIT kinase inhibitors and

primary resistance

5. Secondary resistance to kinase

inhibitory drugs

6. Clinical trials of KIT inhibitors

7. New approaches to treatment

of cancers driven by mutant

KIT

8. Conclusion

9. Expert opinion

Review

Therapeutic targeting of c-KITin cancerLeonie K Ashman† & Renate Griffith†School of Biomedical Sciences & Pharmacy, University of Newcastle and Hunter Medical Research

Institute, Newcastle, Australia

Introduction: Mutated forms of the receptor tyrosine kinase c-KIT are

“drivers” in several cancers and are attractive targets for therapy. While

benefits have been obtained from use of inhibitors of KIT kinase activity

such as imatinib, especially in gastrointestinal stromal tumours (GIST), primary

resistance occurs with certain oncogenic mutations. Furthermore, resistance

frequently develops due to secondary mutations. Approaches to addressing

both of these issues as well as combination therapies to optimise use of KIT

kinase inhibitors are discussed.

Areas covered: This review covers the occurrence of oncogenic KIT mutations

in different cancers and the molecular basis of their action. The action of KIT

kinase inhibitors, especially imatinib, sunitinib, dasatinib and PKC412, on

different primary and secondary mutants is discussed. Outcomes of clinical

trials in GIST, acute myeloid leukaemia (AML), systemic mastocytosis and

melanoma and their implications for future directions are considered.

Expert opinion: Analysis of KIT mutations in individual patients is an essential

prerequisite to the use of kinase inhibitors for therapy, and monitoring for

development of secondary mutations that confer drug resistance is necessary.

However, it is unlikely that KIT inhibitors alone can lead to cure. KIT muta-

tions alone do not seem to be sufficient for transformation; thus identifi-

cation and co-targeting of synergistic oncogenic pathways should lead to

improved outcomes.

Keywords: acute myeloid leukaemia, c-KIT mutations, dasatinib, drug resistance, FTY720,

gastrointestinal stromal tumour, imatinib, kinase inhibitors, mastocytosis, PKC412, sunitinib

Expert Opin. Investig. Drugs (2013) 22(1):103-115

1. Introduction

The receptor tyrosine kinase (RTK), c-KIT, and other type III RTKs, PDGF recep-tor (PDGFR), Colony-stimulating factor-1 receptor (c-FMS) and FLT3 (Fms-liketyrosine kinase receptor-3) play important roles in cancer. KIT was first identifiedas a retroviral oncogene, as the product of the “white spotting” (W) locus in mice,and at the protein level, as a marker of human acute myeloid leukaemia (AML)and normal haemopoietic progenitor cells (reviewed [1]). The phenotype of the Wmouse (characterised by white spotting of the coat, anaemia, lack of mast cellsand infertility) and many subsequent studies on human tissues demonstrated thekey functional role of KIT and its ligand, Stem Cell Factor (SCF) in haemopoiesis,melanogenesis and fertility (especially spermatogenesis). More recently KIT wasshown to be necessary for the development and function of gut pacemaker cells,the Interstitial Cells of Cajal (ICC). This pattern of c-KIT expression and functionhas predicted the cancers in which KIT abnormalities, typically mutations leadingto constitutive activation of the intrinsic kinase, are crucially involved (reviewed [2]).

Typical of the Type III RTK family, the KIT protein consists of an extracellularregion made up of five immunoglobulin (Ig)-like domains, a single transmembranedomain, and an intracellular region including a split kinase domain and

10.1517/13543784.2013.740010 © 2013 Informa UK, Ltd. ISSN 1354-3784, e-ISSN 1744-7658 103All rights reserved: reproduction in whole or in part not permitted

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auto-regulatory domains. Activating mutation of KIT wasfirst reported in the human mast cell leukaemia line,HMC-1 [3]. Two mutations in the same allele, leading toamino acid substitutions V560G and D816V in the juxta-membrane auto-regulatory domain and the kinase domain,respectively, were reported. Each mutation was capable ofcausing ligand-independent KIT activation as demonstratedby tyrosine auto-phosphorylation and promotion of factor-independent growth of murine Ba/F3 cells. Activating muta-tions of KIT are now known to occur in almost all cases ofsystemic mastocytosis (SM) and are often present in otherhaemopoietic lineages in these patients indicating that thetarget may be in the stem cell compartment [4].The most striking involvement of KIT is in gastrointestinal

stromal tumours (GIST) which are derived from the ICC.Activating mutations of KIT in GIST were first reported byHirota and co-workers [5]. It is now known that more than80% of cases display KIT mutations resulting in constitutiveactivation, while a smaller proportion (~ 6%) of cases havemutations in the closely related PDGFR [6]. Use of KITkinase inhibitors has provided a paradigm shift in treatmentof these cancers.In AML KIT mutation is relatively uncommon and is

confined to Core Binding Factor (CBF) leukaemias whichcomprise around 17% of AML and are characterised byt(8;21) or inv(16) [7]. These translocations/inversions result infusion genes such AML-ETO and CBFB-MYHII, respectively,leading to impaired differentiation, while subsequent KITmutation provides a proliferative and survival advantage [8,9].Since KIT expression is down-regulated during normal myelo-poiesis, any disease resulting from activating mutation of KITmight be self-limiting in the absence of a differentiation block.This is in contrast with the mast cell lineage where KIT conti-nues to be highly expressed in mature cells. Recent large studiesin CBF AML indicate that around 37% of adult cases and 19%

of paediatric cases had KIT mutations [10,11]. A higher propor-tion of non-CBF AML cases (~ 30% of all AML) displayactivating mutations in the closely related RTK, FLT3.

As predicted, activating KIT mutations have also beenobserved in germ cell cancers. In testicular seminomasfrequencies of up to 26% have been reported [12]. KIT muta-tions also occur in about 30% of cases of unilateral ovariandysgerminomas but not other histological types [13].

Activating KIT mutations and amplifications have beendemonstrated in melanoma at relatively low frequency(approximately 5%) and mostly in those occurring at accral,mucosal or chronic sun damaged sites [14]. A much largerproportion of melanomas have mutations in B-Raf, which isa downstream effector of KIT and would provide a similarproliferative stimulus [15]. Interestingly, earlier studies indi-cated that, in the majority of melanomas, KIT expression islost on progression [16] indicating distinct roles for KIT indifferent types of melanoma.

While KIT is now known to also be expressed in othertissues, for example, vascular endothelial cells, astrocytes,renal tubules, breast glandular epithelial cells and sweatglands, recurrent mutations have not been described in corre-sponding cancers and early studies have generally failed toshow efficacy of KIT inhibitors in these cancers. This is inaccord with the concept that the target must be a “driver” ofthe cancer (e.g., as evidenced by mutation or over-expression)to be a useful therapeutic target.

2. Mechanism of oncogenic KIT activation

2.1 OverviewThe domain structure of KIT and the location of some com-mon mutations are illustrated in Figure 1. In the normal courseof events ligand binding triggers receptor dimerisation, relief ofauto-inhibitory interactions and trans-phosphorylation withindimer pairs followed by recruitment, phosphorylation andactivation of downstream signalling proteins. There are multi-ple sites of KIT mutation in cancers, with some “hot-spots”corresponding to the intracellular and extracellular juxtamem-brane domains (exons 8, 9 and 11) and the activation loop ofthe kinase domain (exon 17) which lead to disruption ofauto-inhibitory mechanisms. It is noteworthy that the commonsites of KIT mutation differ markedly between cancers. Thismay reflect differential effects of the various mutations ondownstream signalling pathways (Section 3).

The extracellular juxtamembrane domain consisting of thefourth and fifth Ig-like loops is involved in correctly orientingreceptor monomers and stabilising dimers induced by bindingof dimeric SCF [17]. Specifically, this region, encoded by exons8 and 9, directly mediates dimer interactions. Exon 8 is animportant mutation site in AML and results in small dele-tions/substitution usually involving D419 [18]. Exon 9 muta-tions, most commonly an AY insertion [19], occur in about10% of GIST cases and may act by a similar mechanism. Themechanisms of KIT activation by exon 11 and 17 mutations

Article highlights.

. The receptor tyrosine kinase c-KIT is mutated in severaltypes of cancer, notably gastrointestinal tumours (GIST),systemic mastocytosis and subsets of acute myeloidleukaemia and melanoma.

. Small molecule kinase inhibitors (SMIs) such as imatinibhave been successfully used in treatment of GIST.

. Some oncogenic KIT mutations prevent binding ofimatinib and other SMIs; mutation analysis of KIT inindividual patients is needed to determine the optimaldrug and dose.

. Drug resistance also arises due to secondary KITmutations resulting in relapse.

. Cancer stem cells appear to be relatively resistant totreatment with KIT inhibitors.

. Combination of kinase inhibitors with agents targetingcell survival mechanisms and/or synergistic signallingpathways offers considerable promise.

This box summarises key points contained in the article.

L. K. Ashman & R. Griffith

104 Expert Opin. Investig. Drugs (2013) 22(1)

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are discussed in Sections 2.2 and 2.4 in the context of GIST andhaematological malignancies, respectively.

2.2 GISTThe major region of KIT mutation in GIST is within exon11 occurring in about 65% of patients [19]. This exon encodesthe intracellular juxtamembrane domain (JMD), a key auto-regulatory domain of RTKs which stabilises the inactive con-formation of the kinase domain in which the “activationloop” is incorrectly positioned for catalysis [20]. Crystal struc-ture analysis of KIT has shown that in the absence of ligandthe JMD folds back into the active site of the kinase(Figure 2) [21]. Several active and inactive conformations ofKIT, like other RTKs, are believed to exist in equilibriumsuch that in the absence of ligand the inactive conformationsare predominant and the receptor has very low basal activity.Receptor dimerisation following ligand binding allows thisbasal activity to bring about trans-phosphorylation of Y568and Y570 residues in the JMD releasing its interaction with

the kinase domain and promoting the fully active confor-mation [20,22]. Disruption of the JMD by mutation is believedto lead to activation of the kinase by removal of this autoinhi-bition. In GIST there are many different exon 11 mutationsincluding point mutations, tandem duplications, deletionsand insertions and, while the precise mechanisms may differ,these are all believed to act by preventing the interactionof the JMD with the active site cleft [6]. As an example,from the crystal structure of autoinhibited KIT (PDB1T45; [21]) it can be seen that the V560G substitution [23,24]

would result in the loss of multiple hydrophobic interactions(e.g., with N787, I789 and F848) required to stabilisebinding of the JMD to the kinase domain.

2.3 MelanomaThe most common KIT mutations found in melanoma areL576P and K642E, encoded within exons 11 and 13, respec-tively [14]. These substitutions have also been reported inGIST. From the crystal structure of autoinhibited KIT [21],

Mastocytosis

AML

GIST

NH2

TRANSMEMBRANE

JUXTAMEMBRANE

TYROSINEKINASE 1

TYROSINEKINASE 2

TAIL

COOH

KINASEINSERT

IG 1

IG 2

IG 3

IG 4

IG 5

cDNA

del D419*;

A502_Y503 duplication;

F522C; A533D*; V5301

V560G;

K642E‡

del K704_N705

del S715

I748T; L773S

D816V; D816Y; D816H; N822K; V825Imultiple¶;

V559I; multiple§

K5091*

del + ins 416-419

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Figure 1. KIT structure and mutations in cancer. The schematic diagram shows KIT protein domains and the corresponding

gene exons as well as representative mutations that have been reported in SM, AML and GIST. For a more complete list of KIT

mutations in disease, see [47].The figure is reproduced from [99] with the publisher’s permission.

*Familial in mastocytosis.zFamilial in GIST.§Includes: del in region K550_E561; K550I; W557R; V559A; V559D; V560D; G565R; Y568C; del D579; F584S.{Includes: R815k; D816F; I817V; ins V815_I817; D820G; E839K.

Therapeutic targeting of c-KIT in cancer

Expert Opin. Investig. Drugs (2013) 22(1) 105

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it is apparent that the side chain of residue K642 forms anH-bond with the backbone of T574 which stabilises the auto-inhibited structure. L576 stabilises a very short helix in theJMD, which would be disrupted upon mutation to a proline.Additionally, the L576 sidechain makes hydrophobic contactswith the aC helix (V643, Y646). As above, mutation of eitherresidue is expected to favour kinase activation.

2.4 Haematological malignanciesIn haemopoietic malignancies, as well as germ cell tumours,the main region of mutation is exon 17 which encodes theactivation loop of the kinase domain. This region undergoesa major conformational shift on kinase activation (Figure 2).In the inactive state the activation loop obstructs access ofsubstrates to the active site and the DFG sequence at itsN-terminal end is incorrectly aligned for its role in catalysis.Relief of JMD autoinhibition results in kinase activation bygreatly favouring conformations in which the activation loopis shifted away from the active site and the DFG motif adoptsa position enabling correct alignment of ATP and catalysis [25].The active conformation may be stabilized by phosphory-lation of Y823 [22,26]. In systemic mastocytosis (SM) the

predominant activation loop mutation results in substitutionof D816 usually by V but sometimes by H or Y. In CBFAML, D816 and N822 substitutions are common activatingmutations [27]. The D residue corresponding to position816 in KIT is highly conserved in RTKs indicating a keyfunctional role and cell line studies have indicated that theloss of D was important, rather than the residue that replacedit. Early molecular modelling work indicated that substitutionof D816 in KIT destabilised intramolecular interactions inthe inactive conformation of the kinase domain and markedlyfavoured the active conformation [28]. More recentlykinetic studies demonstrated that kinase domain activationis strikingly enhanced in D816H mutant KIT compared toWT [29]. This group showed that the D816H substitutionalso destabilises the interaction of the JMD with thekinase domain.

3. Signalling pathways downstream of KIT

3.1 Signalling by WT KITOnce activated, KIT autophosphorylates on multiple Y resi-dues which serve as docking sites for downstream effectors.

kinase insert

kinase insert

alpha C helix

alpha C helix

JMDJMD

ADP

hinge loophinge loop

Phe811Phe811

activation loop

activation loop

Figure 2. Comparison between the autoinhibited, inactive (left panel), and the active conformation (right panel) of the KIT

kinase. The backbone of the two proteins is shown as a coloured ribbon (gold for the autoinhibited and green for the active

conformation) with key kinase domains highlighted and labelled. The activation loop (purple) clearly takes up very different

positions in the two conformations. All atoms of the residues of the DFG motif at the start of the activation loop have been

displayed as purple sticks, with Phe811 labelled, illustrating the orientation enabling correct alignment of ATP and catalysis in

the active conformation (right panel). The JMD (yellow) is folded back into the kinase domain in the autoinhibited

conformation (left panel) and contacts the rest of the kinase domain, particularly the aC helix (yellow). In the active

conformation, the JMD is folded out and is highly flexible, and thus mostly not resolved (right panel). The kinase insert

(yellow) is a flexible loop connecting the N and C terminal lobes of KIT and other Type III RTKs. This region is not present in

the active conformation crystal structure (right panel). The figure was constructed in Discovery Studio 3.5 (accelrys.com) by

superimposition of two crystal structures: PDB 1T45 (autoinhibited KIT) and PDB 1PKG (KIT in complex with ADP as an ATP

mimic). Superimposition was performed using 6 tethers (Y609, T619, H650, L656, E671, I805). These tethers have been

determined as conformationally invariant using Difference Distance Matrices (DDMs; [100]).



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Several pathways act downstream of KIT to affect cell survivaland proliferation [30]. Src family kinases (SFK), the p85 sub-unit of phosphatidylinositol 3-kinase (PI3K), phospholipaseC-gamma and adaptors that lead to activation of MAP kinasepathways are directly recruited and activated by binding tophospho-Y residues on the receptor.

3.2 Signalling by mutant KITThe different types of KIT mutation associated with particu-lar cancers may be a consequence of their differential effectson signalling pathways downstream of KIT- and/or tissue-specific requirements for particular pathways. Mouse knock-in experiments, in which the wild type Kit alleles werereplaced by alleles coding for receptor lacking recruitmentsites for particular downstream effectors, resulted in tissue-selective consequences (e.g., [31]). It has also been shown thatvarious mutations have differential effects on the activationof signalling pathways downstream of KIT. For example,the MAPK pathway is activated by WT KIT following SCFstimulation, and by mutant KIT in primary GISTs and celllines [32], but not in AML cells or immortalised early myeloidcells expressing D816 mutant KIT where PI3K and/or STAT3 activation appear to be key effectors [33,34]. In con-trast, STAT3 is not activated by WT KIT signalling [34].Whereas WT KIT is mostly located on the cell surface, theD816 mutant is largely in intracellular compartments [35]

which may account for its ability to activate STAT3. Othershave shown that STAT3 activation by another RTK, c-Met,depends on trafficking to an endosomal compartment [36].Recruitment and activation of SFK are of central importancein KIT signalling, but this requirement is overcome by theD816V mutation which confers a Src-like substrate specificityon KIT itself [37]. Alteration of substrate specificity by thecorresponding mutation in murine Kit, D814V, was alsoreported [38].

4. KIT kinase inhibitors and primaryresistance

4.1 Overview -- imatinib resistanceInhibition of the tyrosine kinase activity of the BCR/ABLoncoprotein by imatinib (Gleevec�) in chronic myeloidleukaemia (CML) has revolutionised the treatment of thisdisease [39]. Although imatinib is highly selective comparedwith other tyrosine kinase inhibitors, it also potently blocksthe activity of KIT and PDGFR, suggesting a potential rolein treatment of cancers driven by mutant forms of these recep-tors. At an early stage of evaluation it was noted that clinicallyrelevant KIT mutants differed greatly in their sensitivity toimatinib. Specifically, substitutions at position D816 in theactivation loop rendered the kinase almost completely resis-tant to the drug at clinically achievable doses [23,40]. This likelyreflects the mutation strongly favouring the active conforma-tion of the KIT kinase domain to which, as in the case ofBCR/ABL, imatinib cannot bind [21,41]. In contrast, an exon

11 mutation resulting in the substitution V560G enhancedsensitivity to imatinib by more than 10-fold [23]. Similarresults were obtained with another JMD mutant [42]. Theseobservations have striking implications for the application ofimatinib in GIST and SM in particular.

4.2 Imatinib in treatment of GISTThe action of imatinib in the treatment of GIST has beenextensively evaluated and it is currently the first line treatmentfor metastatic disease and in an adjuvant setting for preventionof relapse of poor prognosis GIST following surgical resection(reviewed [43,44]). As outlined above, more than 80% of GISTshave activating mutations in KIT and a further ~ 5% havemutations in PDGFR. When patients with metastatic diseasewere treated with imatinib (400 mg/day) striking responseswere observed with increased relapse-free or progression-free survival (discussed further in Section 6.1). An interestingdifference was noted between patients with KIT mutationsin exon 11 (~ 65%) and those with mutations in exon9 (~ 10%), with the former group having stronger, moredurable responses [19]. Responses of the exon 9 mutant groupcould be improved by increasing the imatinib dose to800 mg/day [45]. These results are likely to reflect the differencein kinase activation mechanisms between the two classes ofmutant described above. Exon 9 mutations affect an early stageof receptor activation and probably act by mimicking theaction of the ligand. Thus they would be expected to respondto imatinib in a similar way to wild-type (WT) KIT. Incontrast, exon 11 mutants probably act, in general, by releasingthe kinase domain from auto-inhibition by the JMD. Impo-rtantly, this autoinhibitory mechanism also interferes withimatinib binding [21]. Hence JMD mutations such as V560Genhance imatinib inhibition and clinical responses. Similarenhancement of kinase inhibition is seen in cells expressingV560G KIT with the imatinib-related second generationinhibitor, nilotinib [24].

4.3 Imatinib in treatment of SM and AMLImatinib has also been evaluated for treatment of SM whichis typically characterised by activation loop mutations inKIT, particularly the D816V substitution [46]. As statedabove, this mutant form of KIT is highly resistant to thedrug and most cases have proved refractory. Imatinib hasalso been tested without success in AML. In some instances,this was probably due to the use of unselected cases sincemost would not have had KIT mutations. CBF leukaemiasfrequently display KIT mutations in exon 8, usually invol-ving D419, or in exon 17, most commonly affectingD816 or N822, but not in exon 11 (reviewed [47]). Noneof these mutant forms display the enhanced imatinib sensi-tivity of exon 11 mutant KIT, however the N822K mutantand those involving D419 have similar sensitivity to WTKIT [18,27], suggesting that detection of CBF AML patientswith these mutations may select a subgroup of patients likelyto respond.



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4.4 Newer KIT inhibitorsTo approach the primary imatinib resistance of D816 mutantKIT that commonly occurs in SM and also in AML, melanomaand germ cell tumours, newer drugs have been evaluated. Theseinclude multi-targeted inhibitors, dasatinib and PKC412, bothof which can bind to the active conformation of the kinasedomain favoured by D816 mutants. Dasatinib is also a potentinhibitor of SFKs which are importantmediators of KIT actions,thus this inhibitor potentially has dual targets of KIT kinaseactivity per se and SFK-mediated responses [48]. However,although dasatinib strongly inhibits D816 mutant KIT in cellline models, its activity on mutant relative to WT KIT stilldepends on the particular amino acid substitution (Y>>F>V) [49]and varies between reports [50,51]. Unfortunately, studies usingdasatinib in SM patients have yielded disappointing results [52].PKC412 is a multi-targeted staurosporin analogue which hasbeen evaluated as a FLT3 inhibitor in AML [53]. It is proposedto bind in the “hinge” region of the KIT active site which ismuch less influenced by conformational changes associatedwith activation. In the absence of crystal structures ofPKC412 in complex with KIT or related kinases, docking intoa homology model of the active FMS kinase has been used topredict the mode of drug binding. It was shown that PKC412could be docked into a model of the active FMS kinase (basedon PDB 1PKG; [26]), however, it could not be docked into aninactive conformation crystal structure of FMS) [54]. The situa-tion is completely analogous for KIT (authors’ unpublisheddata), where we have not been able to dock PKC412 into eitherof the two inactive conformation crystal structures, whereas itcan be docked into the active conformation of KIT (PDB1PKG). PKC412 is an effective inhibitor of D816V mutantKIT [24,55] and has shown promise in treatment of SM [56].

5. Secondary resistance to kinaseinhibitory drugs

5.1 Resistance due to secondary KIT mutation in

drug-binding residuesSimilar to the case of CML, resistance to imatinib arises ininitially responsive GIST patients treated with the drug,and as with CML, this is almost always due to a secondarymutation in the same KIT allele as the original mutation [57].Secondary mutations identified in this way involve a limitednumber of sites. The most common are point mutations inexons 13 and 14 (encoding the N-terminal lobe of the kinasedomain) resulting in amino acid substitutions V654A orT670I, respectively, which confer imatinib resistance byinterfering with drug binding [58,59]. Residue T670, analo-gous to T315 in BCR/ABL, is the “gatekeeper” residuelocated at the entrance to the hinge region of the ATPbinding cleft. Imatinib forms an H-bond with T670 andthis is lost on mutation. Furthermore, the presence of abulky substituent, either naturally as in the case of FLT3,or through mutation as in T670I KIT, obstructs bindingof imatinib and related drugs such as nilotinib, and alsodasatinib conferring resistance. Residue V654 is directlyinvolved in imatinib binding and replacement with thesmaller A residue results in loss of this hydrophobic interac-tion [59]. To counter imatinib insensitivity due to the“gatekeeper” mutations, the multi-targeted kinase inhibitorsunitinib was developed. This compound, which does notextend beyond the gatekeeper into the catalytic region(Figure 3) is a potent inhibitor of the T670I and V654AKIT mutants. Sunitinib is approved for treatment of GISTfollowing relapse on imatinib [60].

Glu640Thr670

Cys673

Figure 3. Relative orientation of a sunitinib fragment (green sticks) and of imatinib (red sticks) bound to the inactive

conformation of the WT KIT kinase domain. Cys673 is a residue in the hinge region, Thr670 is the “gatekeeper”, and Glu640 is

situated in the aC helix. Imatinib forms hydrogen bonds with Cys673, Thr670, Glu640 and Asp810 (not labelled). The figure

was constructed in Discovery Studio 3.5 (accelrys.com) by superimposition of two crystal structures: PDB 1T46 (KIT in complex

with imatinib) and PDB 3G0E (KIT in complex with sunitinib). The two protein structures are virtually identical, and only the

1T46 protein is shown as a gold ribbon, with selected amino acids displayed as gold sticks and labelled. For clarity, two loops

are also hidden (amino acids 590 -- 603 and 648 -- 656). Sunitinib is not completely resolved in the 3G0E crystal structure, the

solvent-exposed “tail” which would point out of the kinase towards the reader in Figure 3 is missing.



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5.2 Resistance due to secondary activation

loop mutationsNeither T670I nor V654A is an activating mutation. In con-trast, other mutations conferring secondary resistance affectthe activation loop of the kinase domain in particular residueD816, D820, N822 or Y823. D816V and N822K are pri-mary activating mutations which may confer secondary imati-nib resistance by mechanisms similar to those responsible forprimary resistance as described above. While the D816Vmutation confers resistance by strongly favouring the activeconformation of the kinase domain, the precise mechanismin the case of N822K is less certain. Although it is clearly anactivating mutation, in the primary context N822K remainssimilar in imatinib sensitivity to WT KIT [27]. When com-bined with a JMD (exon 11) mutation, the resultant doublemutant also displays similar imatinib sensitivity to WT KIT,however substantially less than the JMD mutant alone(author’s unpublished data). Sunitinib, like imatinib, selec-tively binds to the inactive conformation of the kinase domainand fails to inhibit D816 mutant forms [29,61].

6. Clinical trials of KIT inhibitors

6.1 GISTPrior to the discovery of frequent KIT mutation in GIST [5]

and the advent of targeted kinase inhibitors, metastaticGIST was refractory to existing therapy and median survivalwas around 18 months. Early clinical trials of imatinib fortreatment of patients with metastatic GIST showed remark-able success. Partial responses or disease stabilisation wereachieved in around 80% of patients with 2 year survival of75 -- 80% [62]. This study provided the basis for FDA approvalof imatinib for treatment of metastatic GIST. Ten percent ofpatients displayed primary resistance (defined as progressionwithin the first 6 months of treatment) the frequency of whichwas related to KIT mutational status (WT>exon 9>exon11) [19]. It was subsequently shown that patients with exon9 mutation responded better to an escalated dose of imati-nib [45]. Recent data indicate a median survival of GISTpatients with advanced disease of 5 years with 34% of patientsalive for > 9 years [63]. At this stage it is unclear what durationof imatinib treatment is necessary, but one study has shownthat discontinuation in responding patients after 3 years wasassociated with rapid progression [64].

Following the success in treating patients with advanceddisease, imatinib has been trialled in an adjuvant context inGIST patients with primary disease following potentiallycurative surgery. A definitive Phase III trial reported byDematteo and co-workers [65] demonstrated significant bene-fit of one year of imatinib therapy post-surgery. This led toFDA approval for adjuvant use of imatinib for patients withhigh risk of recurrence (based on tumour size, location,mitotic index, bleeding or rupture). Several other trials arecurrently underway (reviewed [43]). Imatinib has also beenevaluated in a neoadjuvant setting in which the drug is given

prior to, as well as post- surgery [66]. It has been proposedthat this approach could lead to tumour de-bulking inadvanced disease allowing subsequent successful surgery.

Despite the success of imatinib therapy, approximately halfof the patients with metastatic disease develop resistancewithin 2 years, and as in the case of BCR/ABL in CML,this is almost always due to a second mutation in the sameallele of KIT as the primary mutation [57]. The nature of thesemutations (which prevent imatinib binding either directly orby influencing the conformation of the binding site) andsecond-generation inhibitors designed to overcome this resis-tance are discussed in the previous section. From a clinicalperspective, an important observation is that different lesions,and sometimes different regions of the same lesion, inimatinib-resistant patients harbour different secondary muta-tions [67,68]. Since no second generation drug is active on all ofthese mutants, this raises a possible need for use of drug com-binations with potential complications due to toxicity anddrug interactions. Sunitinib was developed as an inhibitor ofmutants with imatinib-resistance due to secondary mutationin the drug binding residues, T670I and V654A of KIT. Ithas proved successful in treating many cases of GIST follo-wing relapse on imatinib and has received FDA approval foruse in this context [60]. However, activation loop mutantsare insensitive to sunitinib [29,69]. Relapse of a patient with atertiary mutation in the activation loop of KIT was reportedduring sunitinib treatment following relapse on imatinibdue to V654A mutation [70]. Other kinase inhibitors, mostlybroad spectrum, are currently being evaluated in clinical trialsin GIST (reviewed [44]).

6.2 SM and AMLIn haemopoietic malignancies mutant forms of KIT have beenobserved in CBF AML and SM. While the majority of AMLpatients achieve remission with aggressive chemotherapy, rela-tively few survive long-term, so that new treatments areurgently needed. Early trials of imatinib in unselected cases ofAML were unsuccessful (e.g., [71]), probably due to the rela-tively low rate of KIT mutations (around 7% overall) and thefrequency of resistant D816 mutations. A small study examinedimatinib responses in three CBF AML cases with KIT muta-tions; two patients with D816 mutation failed to respond whilethe third with an exon 8 mutation showed clinical benefit [72].Because of its low toxicity, imatinib warrants further investi-gation in patients with KIT exon 8 or N822K mutationswhich are relatively common in CBF AML and sensitive tothe drug [18,27]. Dasatinib, which has higher activity onD816 mutant KIT, is currently being trialled in AML(http://clinicaltrials.gov), for example in combination withinduction chemotherapy in AML (NCT01238211) and inmaintenance therapy in CBF AML (NCT00850382).PKC412 (midostaurin), which is currently under evaluationfor treatment of FLT3 mutant AML [53], is also a very goodinhibitor of D816V mutant KIT and has potential fortreatment of cases of AML with this mutant as driver.



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http://clinicaltrials.gov

In SM, KIT mutation (usually D816V) has been observedin both indolent (ISM) and aggressive (ASM) forms of thedisease. ISM is a chronic disease which is treated for symp-tomatic relief, whereas ASM is a debilitating and incurabledisease with shortened life expectancy [46,52] and is a candidatefor therapy with KIT inhibitors. Treatment with imatinib hasgenerally failed to deliver benefit [73,74], likely reflectingits lack of activity on D816 mutant KIT. While dasatinibhas reasonable activity on this mutant in cell lines, resultsin clinical studies have been disappointing with limitedresponses [75,76]. PKC412 is a good inhibitor of D816 mutantKIT and was shown to have activity in one SM patient [56]. Ina subsequent Phase II trial conducted by this group, it showedconsiderable efficacy, but at the expense of substantial toxi-city. This and other ongoing studies have been recentlyreviewed [52,77].

6.3 MelanomaMelanomas commonly express KIT and, since treatmentsof metastatic melanoma prior to the advent of inhibitors ofmutant B-Raf had very poor response rates, early studieswere conducted as to the effect of imatinib in this disease.These studies on unselected patients failed to demonstratesignificant benefit. Subsequently a subset of melanoma cases,mostly those with accral or mucosal phenotype, and lackingB-Raf or K-Ras mutations, have been shown to have acti-vating KIT mutations, most commonly L576P (exon 11)or K642E (exon 13) (e.g., [78]). Two Phase II trials of imati-nib in metastatic melanoma with activating KIT mutationswere reported [78,79] recently, both showing significant bene-fit from drug treatment. In one study, responses occurredacross the spectrum of mutations [79], while in the otherresponses were restricted to patients with L576P or K642Emutations [78]. The results for cases with L576P mutationwere surprising since in vitro studies indicated that thismutant has low sensitivity to imatinib [80,81]. Two patientswith melanoma harbouring L576-mutated KIT weresuccessfully treated with dasatinib, which may have beendue to its activity on SFK as well as or instead of KIT [81].In a Phase II study, dasatinib was shown to have minorbenefit for treatment of metastatic melanoma and to causesubstantial toxicity [82]. It is possible that imatinib or otherKIT inhibitors may have a role in treatment of melanomasdriven by KIT mutations, but a much fuller evaluationis required.

7. New approaches to treatment of cancersdriven by mutant KIT

7.1 Cancer stem cells and resistance to inhibitor

therapyImatinib has profoundly improved the outlook for GISTpatients, but the question remains as to whether this cancercan ever be eradicated with kinase inhibitors alone. Livetumour cells persist in patients treated with imatinib, and

patients rapidly relapse on drug withdrawal [64]. A similarsituation occurs with targeting BCR/ABL in CML wherecomplete elimination of the disease may fail due to the insen-sitivity of the leukaemic stem cell pool to kinase inhibitors [39]

and much can be learned from that experience. A study usinga K641E knock-in mouse model system indicated that normalICC progenitors express low levels of c-Kit. Neither they northeir spontaneously transformed variants were sensitive toimatinib [83]. The authors suggest that cancer stem cell drugs,possibly combined with imatinib, might target these cells.

7.2 Targeting synergising oncogenesFrom the above and other evidence, it follows that develop-ment of overt malignancy requires additional “hits” as wellas mutant KIT and targeting these together with KIT is likelyto be a successful therapeutic approach. Occult GIST“tumorlets” are frequently found in the stomachs of olderindividuals at autopsy. These have typical KIT mutationsseen in GIST, but have benign characteristics [84,85]. Theseobservations indicate that KIT mutation is an early but insuf-ficient event in GIST development, consistent with observa-tions of familial GIST. Recently a key role for the ETSfamily transcription factor ETV-1 acting in synergy withmutant KIT was reported in GIST [86]. Similarly, in CBFAML mutant KIT requires the co-expression of transcriptionfactor fusion proteins that promote survival and block differ-entiation [8,9]. In SM co-operating oncogenic stimuli alsoappear to be necessary for transformation. The presenceof cells expressing the D816V KIT mutant in the blood ofhealthy individuals and in patients with indolent SM hasbeen reported [46,87].

Gleixner and co-workers [88] recently reported that KIT-independent activation of SFKs, Btk and Lyn, synergiseswith D816V KIT in HMC-1 leukaemic mast cells. Growthof HMC-1 cells could be blocked by the synergistic actionof dasatinib, which inhibits both SFK and D816V KIT, andbosutinib which inhibits SFK but not KIT. Similarly, inhibi-tion of Btk and Lyn with low-dose dasatinib (suboptimal forD816V KIT inhibition) combined with PKC412 (whicheffectively inhibits D816V), or with bosutinib and PKC412,blocked HMC-1 cell growth [50,88]. Targeting synergisingoncogenes with small molecule inhibitors appears to be apromising approach although some of these inhibitors havea broad activity spectrum and combinations may causeunacceptable toxicity.

Studies of GIST cell lines indicate that many cells becomequiescent rather than undergoing apoptosis when treated withimatinib and can resume proliferation when the drug is with-drawn [89]. Tumour cells commonly have upregulated survivalmechanisms which are required for the cells to cope with thestress induced by oncogenic disruptions to cell signalling.Targeting mediators of cell survival is being explored as atherapeutic approach. In GIST cell lines apoptosis could beinduced with antagonists to the upregulated BCL2 anti-apoptotic protein [90], while reversing down-regulation of



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pro-apoptotic BIM could bring about cell death [91]. Auto-phagy was shown to be another mechanism by which GISTcell lines avoid death on treatment with KIT inhibitors [89].Treatment with lysosome-targeting agents such as antima-larials chloroquine or quinacrine synergised with imatinib inblocking survival and growth of GIST cells in vitro.

7.3 New approaches to KIT inhibitionA major problem with the use of KIT inhibitors is eitherprimary or secondary resistance due to D816 mutations andthe lack of good drugs to target them. The success of imatiniband sunitinib in GIST is largely due to the scarcity of thesemutants, in contrast to SM in particular. To inhibit D816mutants, a drug needs to bind to the active conformation ofthe kinase which is more structurally constrained than theinactive conformation to which drugs like imatinib and suni-tinib bind. Drugs that bind to the active conformation, suchas dasatinib and PKC412 are, in general, broad spectrumkinase inhibitors with correspondingly greater toxicity. Whilea large number of new small molecule kinase inhibitors areunder evaluation for KIT inhibition (reviewed [44]), experi-ence with dasatinib and PKC412 suggests that they will notbe effective as single agents in patients whose cancers haveD816 KIT mutations. An alternate or additional approachmay be to target the stability of the mutant KIT protein.Fumo et al. [92] showed that the chaperone protein,Hsp90 binds to mutant forms of KIT (including D816V) inHMC-1 mast cell leukaemia lines. Treatment of the cellswith the small molecule Hsp90 inhibitor 17-AAG led to deg-radation of KIT, inhibition of its signalling and promoted celldeath. More recently, IPI-504, a 17-AAG derivative withgreater solubility and improved pharmacological characteris-tics, has been tested in xenograft models of GIST with exon11 or 13 KIT mutations [93]. The drug was shown to causeKIT degradation and block cell proliferation in the tumours,as well as inducing tumour necrosis and shrinkage. Further-more, it acted synergistically with imatinib or sunitinib. How-ever, higher doses, especially in combination with the kinaseinhibitor, caused liver toxicity. It remains to be seen whetherthis will prove to be limiting in humans. Hsp90 inhibitorsare currently being evaluated in Phase II clinical trials inGIST [44].

7.4 Targeting KIT downstream signallingFinally, it may be possible to target downstream signallingpathways that are required for KIT-dependent growth. Forexample, in haemopoetic cells transformed by D816V mutantKIT, PI3K is constitutively and potently activated. Pharmaco-logical inhibition of PI3K caused death of these cells in vitroand mutation of the major KIT recruitment site for PI3K(Y721F) blocked tumourigenicity in syngeneic mice in vivo[33]. Similarly this pathway appears to be critical in survivalof GIST [94]. Several agents targeting PI3K or its downstreameffectors AKT and mTOR are currently under evaluation [95].In haemopoietic cell line models and CBF AML patients the

serine/threonine protein phosphatase PP2A, a known tumoursuppressor, is strikingly down-regulated [96]. This phosphataseis a key regulator of multiple KIT signalling pathways.Pharmacological reactivation of PP2A with FTY720 (a non-toxic drug recently FDA-approved for treatment of multiplesclerosis because of its immunosuppressive action) reducedproliferation and induced apoptosis of murine early myeloidcells expressing D816V KIT in vitro and retarded theirgrowth in syngeneic mice [96]. FTY720 also induced apoptosisin AML patient blasts harbouring D816V/Y KIT [96,97]. Thisapproach offers particular promise in cancers involvingD816 mutations which are highly resistant to commonlyused KIT kinase inhibitors.

8. Conclusion

Mutant forms of KIT are drivers of several cancers includingGIST, SM and subsets of AML, melanoma and testicularseminomas. Multiple different mutations are responsible forconstitutive activity of KIT in a tumour-selective fashion.The small molecule inhibitor, imatinib, has had great benefitin treatment of GIST, but so far kinase inhibitors havemade little impact in SM or other malignancies. This isdue, in part, to the lack of well-validated inhibitors offorms of KIT with certain activation loop mutations. InGIST, treatment with imatinib results in disease control butnot eradication and drug resistance frequently develops dueto secondary mutation in KIT resulting in loss of drugbinding. While new drugs such as sunitinib are effectiveon the most common of these, this drug is not effective onactivation loop mutations. In relapsing GIST patients, differ-ent tumour foci frequently contain different secondary muta-tions such that no single drug is likely to be effective. Atpresent there is no known inhibitor that is active on allobserved KIT secondary mutants. Meanwhile, considerableprogress is being made on identifying pathways that act syner-gistically with KIT to transform cells. Co-targeting thesepathways may lead to tumour control (or even eradication)while being well-tolerated by normal cells, a concept knownas “synthetic lethality” [98]. A particular challenge is to targettumour stem cells which seem to be especially refractory toKIT inhibition.

9. Expert opinion

The observation that different oncogenic mutant forms ofKIT vary greatly in sensitivity to imatinib illustrated theneed for mutation analysis in individual patients in orderto determine which patients would benefit from treatmentwith the drug as well as the appropriate dose to use. This isparticularly true in the case of GIST and CBF AML whichare heterogeneous with respect to the presence and type ofKIT mutations with different inhibitor sensitivity. Mutationanalysis of key exons of KIT is now technically straightfor-ward, relatively inexpensive and is economically justified in



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view of the high cost of the drugs as well as patient benefit.The development of secondary and tertiary resistancemutations in GIST has required development of newsecond-line drugs, but currently there remains a lack ofgood inhibitors for some mutant forms of KIT. Even whereeffective inhibition of KIT is achieved in GIST, diseaseeradication does not occur. There are several exciting candi-dates which could be targeted together with KIT, especiallyin the context of more rapid and extensive reductionof tumour burden. These include modifiers of mutant KITdegradation, cell death or autophagy, and of signallingpathways required for KIT-mediated transformation, forexample, the PI3K pathway and PP2A. Application of com-bination therapies during initial disease treatment shouldlead to optimum tumour clearance and correspondinglyreduce the problem of drug-resistant relapse. However, fordisease cure, eradication of tumour stem cells is necessaryand is not achieved by current therapies. To meet this objec-tive, it is likely that co-operating oncogenes will need to be

targeted together with KIT. Important candidates havebeen identified in GIST and CBF AML.

Acknowledgements

The authors wish to thank NM Verrills, School of BiomedicalSciences & Pharmacy, University of Newcastle for helpfulcomments on the manuscript.

Declaration of interest

Salary and infrastructure support for this work was from aPrincipal Research Fellowship of the National Health &Medical Research Council of Australia and the Universityof Newcastle (LKA) and the University of New South Wales(RG). LKA was a participant in Novartis research panels in2001, 2006 and 2007 and received research funding fromNovartis Australia in 2008. RG has no competing intereststo declare.

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AffiliationLeonie K Ashman†1,2 & Renate Griffith3

†Author for correspondence1University of Newcastle,

New South Wales, Australia2School of Biomedical Sciences & Pharmacy,

University of Newcastle,

University Drive,

Callaghan NSW 2308, Australia

E-mail: [email protected] of Medical Sciences,

University of New South Wales,

Sydney NSW 2052, Australia

E-mail: [email protected]



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