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Posted by on May 14, 2013 in Uncategorized |

c-KIT

Not only are cells expressing
mutated Kit believed to have enhanced proliferation
on exposure to stem cell factor, the ligand of Kit,5,7–10 but
also to exhibit enhanced chemotaxis to stem cell factor.11

 

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

Therapeutic targeting of c-KIT in cancer
Leonie 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 mutations alone do not seem to be sufficient for transformation; thus identification 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. IntroductionThe receptor tyrosine kinase (RTK), c-KIT, and other type III RTKs, PDGF receptor

(PDGFR), Colony-stimulating factor-1 receptor (c-FMS) and FLT3 (Fms-like tyrosine kinase receptor-3) play important roles in cancer. KIT was first identified as 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 W mouse (characterised by white spotting of the coat, anaemia, lack of mast cells and infertility) and many subsequent studies on human tissues demonstrated the key functional role of KIT and its ligand, Stem Cell Factor (SCF) in haemopoiesis, melanogenesis and fertility (especially spermatogenesis). More recently KIT was shown 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 function has predicted the cancers in which KIT abnormalities, typically mutations leading to constitutive activation of the intrinsic kinase, are crucially involved (reviewed [2]). Typical of the Type III RTK family, the KIT protein consists of an extracellular region made up of five immunoglobulin (Ig)-like domains, a single transmembrane domain, and an intracellular region including a split kinase domain and auto-regulatory domains. Activating mutation of KIT was first reported in the human mast cell leukaemia line, HMC-1 [3]. Two mutations in the same allele, leading to amino acid substitutions V560G and D816V in the juxtamembrane auto-regulatory domain and the kinase domain, respectively, were reported. Each mutation was capable of causing ligand-independent KIT activation as demonstrated by tyrosine auto-phosphorylation and promotion of factor independent growth of murine Ba/F3 cells. Activating mutations of KIT are now known to occur in almost all cases of systemic mastocytosis (SM) and are often present in other haemopoietic lineages in these patients indicating that the target 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 by Hirota and co-workers [5]. It is now known that more than 80% of cases display KIT mutations resulting in constitutive
activation, while a smaller proportion (~ 6%) of cases have mutations in the closely related PDGFR [6]. Use of KIT kinase inhibitors has provided a paradigm shift in treatment
of these cancers.
In AML KIT mutation is relatively uncommon and is confined to Core Binding Factor (CBF) leukaemias which comprise around 17% of AML and are characterised by t(8;21) or inv(16) [7]. These translocations/inversions result in fusion genes such AML-ETO and CBFB-MYHII, respectively, leading to impaired differentiation, while subsequent KIT mutation provides a proliferative and survival advantage [8,9].

Since KIT expression is down-regulated during normal myelopoiesis, any disease resulting from activating mutation of KIT might be self-limiting in the absence of a differentiation block. This is in contrast with the mast cell lineage where KIT continues to be highly expressed in mature cells. Recent large studies in CBF AML indicate that around 37% of adult cases and 19% of paediatric cases had KIT mutations [10,11]. A higher proportion of non-CBF AML cases (~ 30% of all AML) display activating mutations in the closely related RTK, FLT3.

As predicted, activating KIT mutations have also been observed in germ cell cancers. In testicular seminomas frequencies of up to 26% have been reported [12]. KIT mutations also occur in about 30% of cases of unilateral ovarian dysgerminomas but not other histological types [13].
Activating KIT mutations and amplifications have been demonstrated in melanoma at relatively low frequency (approximately 5%) and mostly in those occurring at accral, mucosal or chronic sun damaged sites [14]. A much larger proportion of melanomas have mutations in B-Raf, which isa downstream effector of KIT and would provide a similar proliferative stimulus [15]. Interestingly, earlier studies indicated that, in the majority of melanomas, KIT expression is lost on progression [16] indicating distinct roles for KIT in different types of melanoma. While KIT is now known to also be expressed in other tissues, for example, vascular endothelial cells, astrocytes, renal tubules, breast glandular epithelial cells and sweat glands, recurrent mutations have not been described in corresponding cancers and early studies have generally failed to show efficacy of KIT inhibitors in these cancers. This is in accord with the concept that the target must be a “driver” of the cancer (e.g., as evidenced by mutation or over-expression) to be a useful therapeutic target.

2. Mechanism of oncogenic KIT activation

2.1 Overview
The domain structure of KIT and the location of some common mutations are illustrated in Figure 1. In the normal course of events ligand binding triggers receptor dimerisation, relief of auto-inhibitory interactions and trans-phosphorylation within dimer pairs followed by recruitment, phosphorylation and activation of downstream signalling proteins. There are multiplesites of KIT mutation in cancers, with some “hot-spots” corresponding to the intracellular and extracellular juxtamembrane domains (exons 8, 9 and 11) and the activation loop of the kinase domain (exon 17) which lead to disruption of auto-inhibitory mechanisms. It is noteworthy that the common sites of KIT mutation differ markedly between cancers. This may reflect differential effects of the various mutations on downstream signalling pathways (Section 3). The extracellular juxtamembrane domain consisting of the fourth and fifth Ig-like loops is involved in correctly orienting receptor monomers and stabilising dimers induced by binding of dimeric SCF [17]. Specifically, this region, encoded by exons 8 and 9, directly mediates dimer interactions. Exon 8 is an important mutation site in AML and results in small deletions/substitution usually involving D419 [18]. Exon 9 mutations, most commonly an AY insertion [19], occur in about 10% of GIST cases and may act by a similar mechanism. The mechanisms of KIT activation by exon 11 and 17 mutations

Article highlights.
. The receptor tyrosine kinase c-KIT is mutated in several
types of cancer, notably gastrointestinal tumours (GIST),
systemic mastocytosis and subsets of acute myeloid
leukaemia and melanoma.
. Small molecule kinase inhibitors (SMIs) such as imatinib
have been successfully used in treatment of GIST.
. Some oncogenic KIT mutations prevent binding of
imatinib and other SMIs; mutation analysis of KIT in
individual patients is needed to determine the optimal
drug and dose.
. Drug resistance also arises due to secondary KIT
mutations resulting in relapse.
. Cancer stem cells appear to be relatively resistant to
treatment with KIT inhibitors.
. Combination of kinase inhibitors with agents targeting
cell survival mechanisms and/or synergistic signalling
pathways 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 and
haematological malignancies, respectively.
2.2 GIST
The major region of KIT mutation in GIST is within exon
11 occurring in about 65% of patients [19]. This exon encodes
the intracellular juxtamembrane domain (JMD), a key autoregulatory
domain of RTKs which stabilises the inactive conformation
of the kinase domain in which the “activation
loop” is incorrectly positioned for catalysis [20]. Crystal structure
analysis of KIT has shown that in the absence of ligand
the JMD folds back into the active site of the kinase
(Figure 2) [21]. Several active and inactive conformations of
KIT, like other RTKs, are believed to exist in equilibrium
such that in the absence of ligand the inactive conformations
are predominant and the receptor has very low basal activity.
Receptor dimerisation following ligand binding allows this
basal activity to bring about trans-phosphorylation of Y568
and Y570 residues in the JMD releasing its interaction with
the kinase domain and promoting the fully active conformation
[20,22]. Disruption of the JMD by mutation is believed
to lead to activation of the kinase by removal of this autoinhibition.
In GIST there are many different exon 11 mutations
including point mutations, tandem duplications, deletions
and insertions and, while the precise mechanisms may differ,
these are all believed to act by preventing the interaction
of the JMD with the active site cleft [6]. As an example,
from the crystal structure of autoinhibited KIT (PDB
1T45; [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 stabilise
binding of the JMD to the kinase domain.
2.3 Melanoma
The most common KIT mutations found in melanoma are
L576P and K642E, encoded within exons 11 and 13, respectively
[14]. These substitutions have also been reported in
GIST. From the crystal structure of autoinhibited KIT [21],
Mastocytosis
AML
GIST
NH2
TRANSMEMBRANE
JUXTAMEMBRANE
TYROSINE
KINASE 1
TYROSINE
KINASE 2
TAIL
COOH
KINASE
INSERT
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; multiple¶; N822K; V825I
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.
z
Familial 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 an
H-bond with the backbone of T574 which stabilises the autoinhibited
structure. L576 stabilises a very short helix in the
JMD, which would be disrupted upon mutation to a proline.
Additionally, the L576 sidechain makes hydrophobic contacts
with the aC helix (V643, Y646). As above, mutation of either
residue is expected to favour kinase activation.
2.4 Haematological malignancies
In haemopoietic malignancies, as well as germ cell tumours,
the main region of mutation is exon 17 which encodes the
activation loop of the kinase domain. This region undergoes
a major conformational shift on kinase activation (Figure 2).
In the inactive state the activation loop obstructs access of
substrates to the active site and the DFG sequence at its
N-terminal end is incorrectly aligned for its role in catalysis.
Relief of JMD autoinhibition results in kinase activation by
greatly favouring conformations in which the activation loop
is shifted away from the active site and the DFG motif adopts
a position enabling correct alignment of ATP and catalysis [25].
The active conformation may be stabilized by phosphorylation
of Y823 [22,26]. In systemic mastocytosis (SM) the
predominant activation loop mutation results in substitution
of D816 usually by V but sometimes by H or Y. In CBF
AML, D816 and N822 substitutions are common activating
mutations [27]. The D residue corresponding to position
816 in KIT is highly conserved in RTKs indicating a key
functional role and cell line studies have indicated that the
loss of D was important, rather than the residue that replaced
it. Early molecular modelling work indicated that substitution
of D816 in KIT destabilised intramolecular interactions in
the inactive conformation of the kinase domain and markedly
favoured the active conformation [28]. More recently
kinetic studies demonstrated that kinase domain activation
is strikingly enhanced in D816H mutant KIT compared to
WT [29]. This group showed that the D816H substitution
also destabilises the interaction of the JMD with the
kinase domain.
3. Signalling pathways downstream of KIT
3.1 Signalling by WT KIT
Once activated, KIT autophosphorylates on multiple Y residues
which serve as docking sites for downstream effectors.
kinase insert
kinase insert
alpha C helix
alpha C helix
JMD
JMD
ADP
hinge loop hinge loop
Phe811
Phe811
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]).

Several pathways act downstream of KIT to affect cell survival
and proliferation [30]. Src family kinases (SFK), the p85 subunit
of phosphatidylinositol 3-kinase (PI3K), phospholipase
C-gamma and adaptors that lead to activation of MAP kinase
pathways are directly recruited and activated by binding to
phospho-Y residues on the receptor.
3.2 Signalling by mutant KIT
The different types of KIT mutation associated with particular
cancers may be a consequence of their differential effects
on signalling pathways downstream of KIT- and/or tissuespecific
requirements for particular pathways. Mouse knockin
experiments, in which the wild type Kit alleles were
replaced by alleles coding for receptor lacking recruitment
sites for particular downstream effectors, resulted in tissueselective
consequences (e.g., [31]). It has also been shown that
various mutations have differential effects on the activation
of signalling pathways downstream of KIT. For example,
the MAPK pathway is activated by WT KIT following SCF
stimulation, and by mutant KIT in primary GISTs and cell
lines [32], but not in AML cells or immortalised early myeloid
cells expressing D816 mutant KIT where PI3K and/
or STAT3 activation appear to be key effectors [33,34]. In contrast,
STAT3 is not activated by WT KIT signalling [34].
Whereas WT KIT is mostly located on the cell surface, the
D816 mutant is largely in intracellular compartments [35]
which may account for its ability to activate STAT3. Others
have 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 importance
in KIT signalling, but this requirement is overcome by the
D816V mutation which confers a Src-like substrate specificity
on KIT itself [37]. Alteration of substrate specificity by the
corresponding mutation in murine Kit, D814V, was also
reported [38].

4. KIT kinase inhibitors and primaryresistance

4.1 Overview — imatinib resistance
Inhibition of the tyrosine kinase activity of the BCR/ABL
oncoprotein by imatinib (Gleevec) in chronic myeloid
leukaemia (CML) has revolutionised the treatment of this
disease [39]. Although imatinib is highly selective compared
with other tyrosine kinase inhibitors, it also potently blocks
the activity of KIT and PDGFR, suggesting a potential role
in treatment of cancers driven by mutant forms of these receptors.
At an early stage of evaluation it was noted that clinically
relevant KIT mutants differed greatly in their sensitivity to
imatinib. Specifically, substitutions at position D816 in the
activation loop rendered the kinase almost completely resistant
to the drug at clinically achievable doses [23,40]. This likely
reflects the mutation strongly favouring the active conformation
of the KIT kinase domain to which, as in the case of
BCR/ABL, imatinib cannot bind [21,41]. In contrast, an exon
11 mutation resulting in the substitution V560G enhanced
sensitivity to imatinib by more than 10-fold [23]. Similar
results were obtained with another JMD mutant [42]. These
observations have striking implications for the application of
imatinib in GIST and SM in particular.

4.2 Imatinib in treatment of GIST
The action of imatinib in the treatment of GIST has been
extensively evaluated and it is currently the first line treatment
for metastatic disease and in an adjuvant setting for prevention
of relapse of poor prognosis GIST following surgical resection
(reviewed [43,44]). As outlined above, more than 80% of GISTs
have activating mutations in KIT and a further ~ 5% have
mutations in PDGFR. When patients with metastatic disease
were treated with imatinib (400 mg/day) striking responses
were observed with increased relapse-free or progressionfree
survival (discussed further in Section 6.1). An interesting
difference was noted between patients with KIT mutations
in exon 11 (~ 65%) and those with mutations in exon
9 (~ 10%), with the former group having stronger, more
durable responses [19]. Responses of the exon 9 mutant group
could be improved by increasing the imatinib dose to
800 mg/day [45]. These results are likely to reflect the difference
in kinase activation mechanisms between the two classes of
mutant described above. Exon 9 mutations affect an early stage
of receptor activation and probably act by mimicking the
action of the ligand. Thus they would be expected to respond
to imatinib in a similar way to wild-type (WT) KIT. In
contrast, exon 11 mutants probably act, in general, by releasing
the kinase domain from auto-inhibition by the JMD. Importantly,
this autoinhibitory mechanism also interferes with
imatinib binding [21]. Hence JMD mutations such as V560G
enhance imatinib inhibition and clinical responses. Similar
enhancement of kinase inhibition is seen in cells expressing
V560G KIT with the imatinib-related second generation
inhibitor, nilotinib [24].

4.3 Imatinib in treatment of SM and AML
Imatinib has also been evaluated for treatment of SM which
is typically characterised by activation loop mutations in
KIT, particularly the D816V substitution [46]. As stated
above, this mutant form of KIT is highly resistant to the
drug and most cases have proved refractory. Imatinib has
also been tested without success in AML. In some instances,
this was probably due to the use of unselected cases since
most would not have had KIT mutations. CBF leukaemias
frequently display KIT mutations in exon 8, usually involving
D419, or in exon 17, most commonly affecting
D816 or N822, but not in exon 11 (reviewed [47]). None
of these mutant forms display the enhanced imatinib sensitivity
of exon 11 mutant KIT, however the N822K mutant
and those involving D419 have similar sensitivity to WT
KIT [18,27], suggesting that detection of CBF AML patients
with these mutations may select a subgroup of patients likely
to respond.
Therapeutic targeting of c-KIT in cancer
Expert Opin. Investig. Drugs (2013) 22(1) 107
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4.4 Newer KIT inhibitors
To approach the primary imatinib resistance of D816 mutant
KIT that commonly occurs in SMand also in AML, melanoma
and germ cell tumours, newer drugs have been evaluated. These
include multi-targeted inhibitors, dasatinib and PKC412, both
of which can bind to the active conformation of the kinase
domain favoured by D816 mutants. Dasatinib is also a potent
inhibitor of SFKs which are importantmediators ofKITactions,
thus this inhibitor potentially has dual targets of KIT kinase
activity per se and SFK-mediated responses [48]. However,
although dasatinib strongly inhibits D816 mutant KIT in cell
line models, its activity on mutant relative to WT KIT still
depends on the particular amino acid substitution (Y>>F>V) [49]
and varies between reports [50,51]. Unfortunately, studies using
dasatinib in SM patients have yielded disappointing results [52].
PKC412 is a multi-targeted staurosporin analogue which has
been evaluated as a FLT3 inhibitor in AML [53]. It is proposed
to bind in the “hinge” region of the KIT active site which is
much less influenced by conformational changes associated
with activation. In the absence of crystal structures of
PKC412 in complex with KIT or related kinases, docking into
a homology model of the active FMS kinase has been used to
predict the mode of drug binding. It was shown that PKC412
could be docked into a model of the active FMS kinase (based
on PDB 1PKG; [26]), however, it could not be docked into an
inactive conformation crystal structure of FMS) [54]. The situation
is completely analogous for KIT (authors’ unpublished
data), where we have not been able to dock PKC412 into either
of the two inactive conformation crystal structures, whereas it
can be docked into the active conformation of KIT (PDB
1PKG). PKC412 is an effective inhibitor of D816V mutant
KIT [24,55] and has shown promise in treatment of SM [56].

5. Secondary resistance to kinase
inhibitory drugs

5.1 Resistance due to secondary KIT mutation in
drug-binding residues
Similar to the case of CML, resistance to imatinib arises in
initially responsive GIST patients treated with the drug,
and as with CML, this is almost always due to a secondary
mutation in the same KIT allele as the original mutation [57].
Secondary mutations identified in this way involve a limited
number of sites. The most common are point mutations in
exons 13 and 14 (encoding the N-terminal lobe of the kinase
domain) resulting in amino acid substitutions V654A or
T670I, respectively, which confer imatinib resistance by
interfering with drug binding [58,59]. Residue T670, analogous
to T315 in BCR/ABL, is the “gatekeeper” residue
located at the entrance to the hinge region of the ATP
binding cleft. Imatinib forms an H-bond with T670 and
this is lost on mutation. Furthermore, the presence of a
bulky substituent, either naturally as in the case of FLT3,
or through mutation as in T670I KIT, obstructs binding
of imatinib and related drugs such as nilotinib, and also
dasatinib conferring resistance. Residue V654 is directly
involved in imatinib binding and replacement with the
smaller A residue results in loss of this hydrophobic interaction
[59]. To counter imatinib insensitivity due to the
“gatekeeper” mutations, the multi-targeted kinase inhibitor
sunitinib was developed. This compound, which does not
extend beyond the gatekeeper into the catalytic region
(Figure 3) is a potent inhibitor of the T670I and V654A
KIT mutants. Sunitinib is approved for treatment of GIST
following relapse on imatinib [60].
Thr670 Glu640
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.

5.2 Resistance due to secondary activation loop mutations
Neither T670I nor V654A is an activating mutation. In contrast,
other mutations conferring secondary resistance affect
the activation loop of the kinase domain in particular residue
D816, D820, N822 or Y823. D816V and N822K are primary
activating mutations which may confer secondary imatinib
resistance by mechanisms similar to those responsible for
primary resistance as described above. While the D816V
mutation confers resistance by strongly favouring the active
conformation of the kinase domain, the precise mechanism
in the case of N822K is less certain. Although it is clearly an
activating mutation, in the primary context N822K remains
similar in imatinib sensitivity to WT KIT [27]. When combined
with a JMD (exon 11) mutation, the resultant double
mutant also displays similar imatinib sensitivity to WT KIT,
however substantially less than the JMD mutant alone
(author’s unpublished data). Sunitinib, like imatinib, selectively
binds to the inactive conformation of the kinase domain
and fails to inhibit D816 mutant forms [29,61].

6. Clinical trials of KIT inhibitors

6.1 GIST
Prior to the discovery of frequent KIT mutation in GIST [5]
and the advent of targeted kinase inhibitors, metastatic
GIST was refractory to existing therapy and median survival
was around 18 months. Early clinical trials of imatinib for
treatment of patients with metastatic GIST showed remarkable
success. Partial responses or disease stabilisation were
achieved in around 80% of patients with 2 year survival of
75 — 80% [62]. This study provided the basis for FDA approval
of imatinib for treatment of metastatic GIST. Ten percent of
patients displayed primary resistance (defined as progression
within the first 6 months of treatment) the frequency of which
was related to KIT mutational status (WT>exon 9>exon
11) [19]. It was subsequently shown that patients with exon
9 mutation responded better to an escalated dose of imatinib
[45]. Recent data indicate a median survival of GIST
patients with advanced disease of 5 years with 34% of patients
alive for > 9 years [63]. At this stage it is unclear what duration
of imatinib treatment is necessary, but one study has shown
that discontinuation in responding patients after 3 years was
associated with rapid progression [64].
Following the success in treating patients with advanced
disease, imatinib has been trialled in an adjuvant context in
GIST patients with primary disease following potentially
curative surgery. A definitive Phase III trial reported by
Dematteo and co-workers [65] demonstrated significant benefit
of one year of imatinib therapy post-surgery. This led to
FDA approval for adjuvant use of imatinib for patients with
high risk of recurrence (based on tumour size, location,
mitotic index, bleeding or rupture). Several other trials are
currently underway (reviewed [43]). Imatinib has also been
evaluated in a neoadjuvant setting in which the drug is given
prior to, as well as post- surgery [66]. It has been proposed
that this approach could lead to tumour de-bulking in
advanced disease allowing subsequent successful surgery.
Despite the success of imatinib therapy, approximately half
of the patients with metastatic disease develop resistance
within 2 years, and as in the case of BCR/ABL in CML,
this is almost always due to a second mutation in the same
allele of KIT as the primary mutation [57]. The nature of these
mutations (which prevent imatinib binding either directly or
by influencing the conformation of the binding site) and
second-generation inhibitors designed to overcome this resistance
are discussed in the previous section. From a clinical
perspective, an important observation is that different lesions,
and sometimes different regions of the same lesion, in
imatinib-resistant patients harbour different secondary mutations
[67,68]. Since no second generation drug is active on all of
these mutants, this raises a possible need for use of drug combinations
with potential complications due to toxicity and
drug interactions. Sunitinib was developed as an inhibitor of
mutants with imatinib-resistance due to secondary mutation
in the drug binding residues, T670I and V654A of KIT. It
has proved successful in treating many cases of GIST following
relapse on imatinib and has received FDA approval for
use in this context [60]. However, activation loop mutants
are insensitive to sunitinib [29,69]. Relapse of a patient with a
tertiary mutation in the activation loop of KIT was reported
during sunitinib treatment following relapse on imatinib
due to V654A mutation [70]. Other kinase inhibitors, mostly
broad spectrum, are currently being evaluated in clinical trials
in GIST (reviewed [44]).

6.2 SM and AML
In haemopoietic malignancies mutant forms of KIT have been
observed in CBF AML and SM. While the majority of AML
patients achieve remission with aggressive chemotherapy, relatively
few survive long-term, so that new treatments are
urgently needed. Early trials of imatinib in unselected cases of
AML were unsuccessful (e.g., [71]), probably due to the relatively
low rate of KIT mutations (around 7% overall) and the
frequency of resistant D816 mutations. A small study examined
imatinib responses in three CBF AML cases with KIT mutations;
two patients with D816 mutation failed to respond while
the third with an exon 8 mutation showed clinical benefit [72].
Because of its low toxicity, imatinib warrants further investigation
in patients with KIT exon 8 or N822K mutations
which are relatively common in CBF AML and sensitive to
the drug [18,27]. Dasatinib, which has higher activity on
D816 mutant KIT, is currently being trialled in AML
(http://clinicaltrials.gov), for example in combination with
induction chemotherapy in AML (NCT01238211) and in
maintenance therapy in CBF AML (NCT00850382).
PKC412 (midostaurin), which is currently under evaluation
for treatment of FLT3 mutant AML [53], is also a very good
inhibitor of D816V mutant KIT and has potential for
treatment of cases of AML with this mutant as driver.
Therapeutic targeting of c-KIT in cancer

In SM, KIT mutation (usually D816V) has been observed
in both indolent (ISM) and aggressive (ASM) forms of the
disease. ISM is a chronic disease which is treated for symptomatic
relief, whereas ASM is a debilitating and incurable
disease with shortened life expectancy [46,52] and is a candidate
for therapy with KIT inhibitors. Treatment with imatinib has
generally failed to deliver benefit [73,74], likely reflecting
its lack of activity on D816 mutant KIT. While dasatinib
has reasonable activity on this mutant in cell lines, results
in clinical studies have been disappointing with limited
responses [75,76]. PKC412 is a good inhibitor of D816 mutant
KIT and was shown to have activity in one SM patient [56]. In
a subsequent Phase II trial conducted by this group, it showed
considerable efficacy, but at the expense of substantial toxicity.
This and other ongoing studies have been recently
reviewed [52,77].

6.3 Melanoma
Melanomas commonly express KIT and, since treatments
of metastatic melanoma prior to the advent of inhibitors of
mutant B-Raf had very poor response rates, early studies
were conducted as to the effect of imatinib in this disease.
These studies on unselected patients failed to demonstrate
significant benefit. Subsequently a subset of melanoma cases,
mostly those with accral or mucosal phenotype, and lacking
B-Raf or K-Ras mutations, have been shown to have activating
KIT mutations, most commonly L576P (exon 11)
or K642E (exon 13) (e.g., [78]). Two Phase II trials of imatinib
in metastatic melanoma with activating KIT mutations
were reported [78,79] recently, both showing significant benefit
from drug treatment. In one study, responses occurred
across the spectrum of mutations [79], while in the other
responses were restricted to patients with L576P or K642E
mutations [78]. The results for cases with L576P mutation
were surprising since in vitro studies indicated that this
mutant has low sensitivity to imatinib [80,81]. Two patients
with melanoma harbouring L576-mutated KIT were
successfully treated with dasatinib, which may have been
due to its activity on SFK as well as or instead of KIT [81].
In a Phase II study, dasatinib was shown to have minor
benefit for treatment of metastatic melanoma and to cause
substantial toxicity [82]. It is possible that imatinib or other
KIT inhibitors may have a role in treatment of melanomas
driven by KIT mutations, but a much fuller evaluation
is required.

7. New approaches to treatment of cancers
driven by mutant KIT

7.1 Cancer stem cells and resistance to inhibitor
therapy
Imatinib has profoundly improved the outlook for GIST
patients, but the question remains as to whether this cancer
can ever be eradicated with kinase inhibitors alone. Live
tumour cells persist in patients treated with imatinib, and
patients rapidly relapse on drug withdrawal [64]. A similar
situation occurs with targeting BCR/ABL in CML where
complete elimination of the disease may fail due to the insensitivity
of the leukaemic stem cell pool to kinase inhibitors [39]
and much can be learned from that experience. A study using
a K641E knock-in mouse model system indicated that normal
ICC progenitors express low levels of c-Kit. Neither they nor
their spontaneously transformed variants were sensitive to
imatinib [83]. The authors suggest that cancer stem cell drugs,
possibly combined with imatinib, might target these cells.

7.2 Targeting synergising oncogenes
From the above and other evidence, it follows that development
of overt malignancy requires additional “hits” as well
as mutant KIT and targeting these together with KIT is likely
to be a successful therapeutic approach. Occult GIST
“tumorlets” are frequently found in the stomachs of older
individuals at autopsy. These have typical KIT mutations
seen in GIST, but have benign characteristics [84,85]. These
observations indicate that KIT mutation is an early but insufficient
event in GIST development, consistent with observations
of familial GIST. Recently a key role for the ETS
family transcription factor ETV-1 acting in synergy with
mutant KIT was reported in GIST [86]. Similarly, in CBF
AML mutant KIT requires the co-expression of transcription
factor fusion proteins that promote survival and block differentiation
[8,9]. In SM co-operating oncogenic stimuli also
appear to be necessary for transformation. The presence
of cells expressing the D816V KIT mutant in the blood of
healthy individuals and in patients with indolent SM has
been reported [46,87].
Gleixner and co-workers [88] recently reported that KITindependent
activation of SFKs, Btk and Lyn, synergises
with D816V KIT in HMC-1 leukaemic mast cells. Growth
of HMC-1 cells could be blocked by the synergistic action
of dasatinib, which inhibits both SFK and D816V KIT, and
bosutinib which inhibits SFK but not KIT. Similarly, inhibition
of Btk and Lyn with low-dose dasatinib (suboptimal for
D816V KIT inhibition) combined with PKC412 (which
effectively inhibits D816V), or with bosutinib and PKC412,
blocked HMC-1 cell growth [50,88]. Targeting synergising
oncogenes with small molecule inhibitors appears to be a
promising approach although some of these inhibitors have
a broad activity spectrum and combinations may cause
unacceptable toxicity.
Studies of GIST cell lines indicate that many cells become
quiescent rather than undergoing apoptosis when treated with
imatinib and can resume proliferation when the drug is withdrawn
[89]. Tumour cells commonly have upregulated survival
mechanisms which are required for the cells to cope with the
stress induced by oncogenic disruptions to cell signalling.
Targeting mediators of cell survival is being explored as a
therapeutic approach. In GIST cell lines apoptosis could be
induced with antagonists to the upregulated BCL2 antiapoptotic
protein [90], while reversing down-regulation of pro-apoptotic BIM could bring about cell death [91]. Autophagy
was shown to be another mechanism by which GIST
cell lines avoid death on treatment with KIT inhibitors [89].
Treatment with lysosome-targeting agents such as antimalarials
chloroquine or quinacrine synergised with imatinib in
blocking survival and growth of GIST cells in vitro.

7.3 New approaches to KIT inhibition
A major problem with the use of KIT inhibitors is either
primary or secondary resistance due to D816 mutations and
the lack of good drugs to target them. The success of imatinib
and sunitinib in GIST is largely due to the scarcity of these
mutants, in contrast to SM in particular. To inhibit D816
mutants, a drug needs to bind to the active conformation of
the kinase which is more structurally constrained than the
inactive conformation to which drugs like imatinib and sunitinib
bind. Drugs that bind to the active conformation, such
as dasatinib and PKC412 are, in general, broad spectrum
kinase inhibitors with correspondingly greater toxicity. While
a large number of new small molecule kinase inhibitors are
under evaluation for KIT inhibition (reviewed [44]), experience
with dasatinib and PKC412 suggests that they will not
be effective as single agents in patients whose cancers have
D816 KIT mutations. An alternate or additional approach
may 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) in
HMC-1 mast cell leukaemia lines. Treatment of the cells
with the small molecule Hsp90 inhibitor 17-AAG led to degradation
of KIT, inhibition of its signalling and promoted cell
death. More recently, IPI-504, a 17-AAG derivative with
greater solubility and improved pharmacological characteristics,
has been tested in xenograft models of GIST with exon
11 or 13 KIT mutations [93]. The drug was shown to cause
KIT degradation and block cell proliferation in the tumours,
as well as inducing tumour necrosis and shrinkage. Furthermore,
it acted synergistically with imatinib or sunitinib. However,
higher doses, especially in combination with the kinase
inhibitor, caused liver toxicity. It remains to be seen whether
this will prove to be limiting in humans. Hsp90 inhibitors
are currently being evaluated in Phase II clinical trials in
GIST [44].

7.4 Targeting KIT downstream signalling
Finally, it may be possible to target downstream signalling
pathways that are required for KIT-dependent growth. For
example, in haemopoetic cells transformed by D816V mutant
KIT, PI3K is constitutively and potently activated. Pharmacological
inhibition of PI3K caused death of these cells in vitro
and 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 survival
of GIST [94]. Several agents targeting PI3K or its downstream
effectors 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 tumour
suppressor, is strikingly down-regulated [96]. This phosphatase
is a key regulator of multiple KIT signalling pathways.
Pharmacological reactivation of PP2A with FTY720 (a nontoxic
drug recently FDA-approved for treatment of multiple
sclerosis because of its immunosuppressive action) reduced
proliferation and induced apoptosis of murine early myeloid
cells expressing D816V KIT in vitro and retarded their
growth in syngeneic mice [96]. FTY720 also induced apoptosis
in AML patient blasts harbouring D816V/Y KIT [96,97]. This
approach offers particular promise in cancers involving
D816 mutations which are highly resistant to commonly
used KIT kinase inhibitors.

8. Conclusion
Mutant forms of KIT are drivers of several cancers including
GIST, SM and subsets of AML, melanoma and testicular
seminomas. Multiple different mutations are responsible for
constitutive activity of KIT in a tumour-selective fashion.
The small molecule inhibitor, imatinib, has had great benefit
in treatment of GIST, but so far kinase inhibitors have
made little impact in SM or other malignancies. This is
due, in part, to the lack of well-validated inhibitors of
forms of KIT with certain activation loop mutations. In
GIST, treatment with imatinib results in disease control but
not eradication and drug resistance frequently develops due
to secondary mutation in KIT resulting in loss of drug
binding. While new drugs such as sunitinib are effective
on the most common of these, this drug is not effective on
activation loop mutations. In relapsing GIST patients, different
tumour foci frequently contain different secondary mutations
such that no single drug is likely to be effective. At
present there is no known inhibitor that is active on all
observed KIT secondary mutants. Meanwhile, considerable
progress is being made on identifying pathways that act synergistically
with KIT to transform cells. Co-targeting these
pathways may lead to tumour control (or even eradication)
while being well-tolerated by normal cells, a concept known
as “synthetic lethality” [98]. A particular challenge is to target
tumour stem cells which seem to be especially refractory to
KIT inhibition.

9. Expert opinion
The observation that different oncogenic mutant forms of
KIT vary greatly in sensitivity to imatinib illustrated the
need for mutation analysis in individual patients in order
to determine which patients would benefit from treatment
with the drug as well as the appropriate dose to use. This is
particularly true in the case of GIST and CBF AML which
are heterogeneous with respect to the presence and type of
KIT mutations with different inhibitor sensitivity. Mutation
analysis of key exons of KIT is now technically straightforward,
relatively inexpensive and is economically justified in
Therapeutic targeting of c-KIT in cancer
Expert Opin. Investig. Drugs (2013) 22(1) 111
Expert Opin. Investig. Drugs Downloaded from informahealthcare.com by University of North Carolina on 03/22/13
For personal use only.
view of the high cost of the drugs as well as patient benefit.
The development of secondary and tertiary resistance
mutations in GIST has required development of new
second-line drugs, but currently there remains a lack of
good inhibitors for some mutant forms of KIT. Even where
effective inhibition of KIT is achieved in GIST, disease
eradication does not occur. There are several exciting candidates
which could be targeted together with KIT, especially
in the context of more rapid and extensive reduction
of tumour burden. These include modifiers of mutant KIT
degradation, cell death or autophagy, and of signalling
pathways required for KIT-mediated transformation, for
example, the PI3K pathway and PP2A. Application of combination
therapies during initial disease treatment should
lead to optimum tumour clearance and correspondingly
reduce the problem of drug-resistant relapse. However, for
disease cure, eradication of tumour stem cells is necessary
and is not achieved by current therapies. To meet this objective,
it is likely that co-operating oncogenes will need to be
targeted together with KIT. Important candidates have
been identified in GIST and CBF AML.

Acknowledgements
The authors wish to thank NM Verrills, School of Biomedical
Sciences & Pharmacy, University of Newcastle for helpful
comments on the manuscript.

Declaration of interest
Salary and infrastructure support for this work was from a
Principal Research Fellowship of the National Health &
Medical Research Council of Australia and the University
of Newcastle (LKA) and the University of New South Wales
(RG). LKA was a participant in Novartis research panels in
2001, 2006 and 2007 and received research funding from
Novartis Australia in 2008. RG has no competing interests
to declare.

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Affiliation
Leonie K Ashman†1,2 & Renate Griffith3
†Author for correspondence
1University of Newcastle,
New South Wales, Australia
2School of Biomedical Sciences & Pharmacy,
University of Newcastle,
University Drive,
Callaghan NSW 2308, Australia
E-mail: leonie.ashman@newcastle.edu.au
3School of Medical Sciences,
University of New South Wales,
Sydney NSW 2052, Australia
E-mail: r.griffith@unsw.edu.au
Therapeutic targeting of c-KIT in cancer
Expert Opin. Investig. Drugs (2013) 22(1) 115
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