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KIT as a Therapeutic Target in Melanoma
PERSPECTIVE
KIT as a Therapeutic Target in Melanoma Maria C. Garrido1 and Boris C. Bastian1,2 Recently, genetic alterations activating the receptor tyrosine kinase KIT have been shown in some types of melanoma. KIT mutations can be successfully targeted by approved drugs in other cancers. Emerging evidence suggests that melanomas with KIT activation may also respond to KIT inhibitors that are already in clinical use. If confirmed in ongoing clinical trials, this experience would underscore the importance of recognizing the biological diversity among melanomas, representing a first decisive step toward the individualized and mechanism-based treatment of melanoma. Journal of Investigative Dermatology (2010) 130, 20–27; doi:10.1038/jid.2009.334; published online 22 October 2009
INTRODUCTION KIT is a member of the type-III transmembrane receptor tyrosine kinase familythat comprises five extracellular immunoglobulin domains, a single transmembrane region, an inhibitory cytoplasmic juxtamembrane domain, and a split cytoplasmic kinase domain separated by a kinase insert segment (Yarden et al., 1987). Under physiological conditions, binding of the KIT ligand, stem-cell factor, to the extracellular domain of the receptor results in receptor dimerization, activation of the intracellular tyrosine kinase domain through autophosphorylation of specific tyrosine residues, and receptor activation (Lev et al., 1992). The downstream signal transduction pathways include the mitogen-activated protein kinase, phosphatidylinositol-30 -kinase, and JAK/STAT (JAK/signal transducers and activators of transcription) pathways. The intracellular signaling through KIT plays a critical role in the development of several mammalian cell types, including melanocytes, hematopoietic progenitor cells, mast cells, primordial germ cells, and interstitial cells of Cajal (Nishikawa et al., 1991; Galli et al., 1995). Although it has been extensively shown that KIT
signaling is essential for melanocyte development, its precise role in migration, survival, proliferation, and differentiation remains incompletely understood. KIT function is important for survival of melanoblasts, as melanocyte precursors with loss-of-function mutations in KIT never disperse and ultimately disappear (Wehrle-Haller and Weston, 1995). In mouse, survival of melanogenic subpopulations of neural crest cells depends on stem-cell factor for a critical period, which begins after the second day of dispersal, and lasts for about 4 days, ending about the time that melanocytes terminally differentiate, as indicated by the presence of functional melanosomes (Morrison-Graham and Weston, 1993). A role for KIT in the dorsoventral migration of melanocytes is also indicated by the fact that loss-of-function mutations in the KIT pathway lead to patterned pigmentation phenotypes such as white midline spotting in animals and piebaldism in humans (Giebel and Spritz, 1991). Specifically, KIT activation has been shown to be transiently required in the dorsal dermatome before the onset of trunk neural crest dispersal on the lateral pathway (Wehrle-Haller and Weston,
1995). Alexeev and Yoon (2006) have shown that activation of the KIT receptor primarily results in stimulation of migration rather than proliferation of melanocytes and found that it is also responsible for morphological changes of melanocytes such as spindle-shaped bodies and reduced number of dendrites (Alexeev and Yoon, 2006). KIT activation in melanocytes results in rapid increase in actin stress-fiber formation and elevated melanocyte migration on fibronectin (Scott et al., 1996), indicating a role for KIT in the reorganization of the cytoskeleton and higher migratory properties of melanocytes. When melanocytes with activating KIT mutations are transplanted into the dorsal skin of albino mice, cells show a distinctive migration pattern, from the injected sites to the upper dermis and dermal–epidermal border, toward the follicular and/or interfollicular keratinocytes (Kunisada et al., 1998). Despite clear evidence that KIT activation is linked to phenotypes such as migration and survival associated with cancer cells, its role as an oncogene melanoma did not immediately become clear. Early studies of KIT in melanoma lesions found its
1
Department of Dermatology and UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA and 2Department of Pathology and UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA Correspondence: Dr Boris C. Bastian, Departments of Dermatology and Pathology, University of California, San Francisco, UCSF Helen Diller Family Comprehensive Cancer Center, Box 0808, San Francisco, California 94143-0808, USA. E-mail: [email protected] Abbreviations: CSD, chronic sun-damaged; GIST, gastrointestinal stromal tumor; PDGFR, platelet-derived growth factor receptor; PI3K, phosphatidylinositol30 -kinase Received 10 April 2009; revised 28 June 2009; accepted 31 July 2009; published online 22 October 2009
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& 2010 The Society for Investigative Dermatology
MC Garrido and BC Bastian KIT as a Therapeutic Target in Melanoma
expression downregulated frequently during progression from early to advanced lesions (Lassam and Bickford, 1992; Natali et al., 1992; Montone et al., 1997), and functional studies even showed that KIT had antiproliferative and antimetastatic properties in some settings (Zakut et al., 1993; Huang et al., 1996). When expressed in melanoma cell lines without KIT expression, KIT induced cell-cycle arrest and apoptosis (Huang et al., 1996). Also, although constitutive activation of KIT in primary melanocytes resulted in increased migration, it also decreased proliferation (Alexeev and Yoon, 2006). These observations have led to the view that KIT may rather act as a tumor suppressor in melanoma, and that to acquire proliferative advantage and escape from the epidermal boundaries, transformed melanocytes need to lose KIT expression. Our attention to KIT as a potential melanoma oncogene was first attracted by a narrow sub-centromeric amplification on chromosome 4q that we observed in acral melanoma, and which appeared to include the KIT locus (Bastian et al., 1998). However, the method of chromosome-based comparative genomic hybridization used at the time did not provide the resolution to sufficiently narrow the affected genomic region, and the published data on the tumor-suppressive effects of KIT at the time appeared to be inconsistent with KIT as a compelling driver gene within the amplicon. Our interest in KIT rose again when we observed a striking field effect in acral melanomas, in which we found single basal melanocytes in the basilar nonlesional epidermis adjacent to acral melanomas that shared similar chromosomal aberrations with the nearby melanoma (Bastian et al., 2000). The extent of these field cells was substantial, reaching up to 2 cm in some cases, suggesting that the melanoma cells were highly migratory, but still under homeostatic control, as they were not increased in number and still equidistantly spaced just as normal melanocytes (North et al., 2008). When BRAF was discovered by the Sanger Center as a melanoma oncogene, we conducted follow-up studies of a larger series of
primary melanomas and found that BRAF mutations were infrequent in mucosal, acral, and chronic sundamaged (CSD) melanomas (Maldonado et al., 2003). The relative dearth of BRAF mutations in these three categories of melanoma, which share a common lentigenous growth pattern characterized by single melanocytes distributed along the basilar epidermis as a common morphological feature, raised the question of what oncogene was activated in these melanoma types, if not BRAF. The lentigenous growth pattern along with the field effect in acral melanoma raised the possibility that a gene inducing melanocyte migration may be involved. A larger study using array comparative genomic hybridization provided us with a refined boundary of the amplicon on chromosome 4q and confirmed that KIT was included in the region, residing at the peak of the amplicon. It was also remarkable that the amplification was virtually exclusive with mutations in BRAF or NRAS (Curtin et al., 2006). As KIT was known to induce melanocyte migration and because of its functional overlap with BRAF and NRAS, KIT rose to the top of the list of candidate genes within the region. When we sequenced KIT, we found recurrent mutations in KIT in mucosal (21%), acral (11%), and CSD melanomas (17%), but not in nonCSD melanomas, in which BRAF mutations predominate. At the time of publication of our results, others had already described occasional mutations of KIT in a few cases of melanomas. Went et al. (2004) performed immunohistochemistry for KIT protein and found expression in 36% (14 of 39). One of the cases with positive expression showed a mutation in exon 11 (L576P). Another study analyzed 100 melanoma metastases not characterized further and showed expression in 29 cases (29%), two of which had mutations, also in exon 11 (L576P) (Willmore-Payne et al., 2005). In addition to KIT mutations, we found frequent copy-number increases of the KIT locus in the same three melanoma categories, which also tended to be mutually exclusive of mutations in BRAF and NRAS. This suggested that copy-number increases of wild-type
KIT may drive these melanomas, or that the copy-number increases at 4q12 implicate another nearby oncogene. The obvious candidate was plateletderived growth factor receptor-A (PDGFRA), a related receptor tyrosine kinase involved in multiple cancer types. However, mutation analysis of PDGFRA in melanomas with and without copy-number increases at 4q12 did not reveal any mutations of PDGFRA. It, therefore, remains plausible that the increased gene dosage of KIT may be pathogenetically relevant in some melanomas. Mutations and copy-number increases in KIT were mostly mutually exclusive. However, mutations at codon K642E were frequently accompanied by amplifications of the mutated allele. This mutation has also been reported in the germ line of patients with familial gastrointestinal stromal tumors, indicating that it may be a weak gain-of-function allele that is tolerable in the germ line (Isozaki et al., 2000), but needs to be increased in gene dosage by amplification to be fully oncogenic. A recent, larger follow-up study has confirmed the recurrent mutations in acral and mucosal melanomas, but did not distinguish between CSD and non-CSD melanomas (Beadling et al., 2008). Table 1 summarizes the mutations reported in KIT to date and their relative distribution across melanoma subtypes. We would like to note that the finding of known oncogenic mutations in KIT in melanoma subsets is not necessarily contradictory to the findings of the tumor-suppressive effects of KIT reported earlier. In fact, the discrepancies between our results and previous reports in the literature (Lassam and Bickford, 1992; Natali et al., 1992; Montone et al., 1997) could be reconciled by the fact that those studies were conducted on melanoma cell lines, which are usually derived from the melanoma subtype characterized by its occurrence on intermittently exposed skin (non-CSD). Together with the dearth of KIT mutations or copy-number increases of KIT in non-CSD melanoma, the divergent findings may, therefore, indicate different roles of KIT signaling in these www.jidonline.org
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MC Garrido and BC Bastian KIT as a Therapeutic Target in Melanoma
Table 1. KIT mutations in melanomas subtypes Reference
Mucosal melanomas
Acral melanomas
CSD melanomas
0/6 (0%)
2/22 (9%)
ND
ND
ND
Ashida et al. (2009) Rivera et al. (2008)
4/18 (22.2%)
Curtin et al. (2006)
8/38 (21%)
3/28 (11%)
3/18 (16.7%)
Satzger et al. (2008)
6/37 (16%)
ND
ND
Antonescu et al. (2007)
3/20 (15%)
Beadling et al. (2008)
7/45 (15.6%)
Carvajal et al. (2009)
12/45 (27%)
ND
ND
3/13 (23%)
1/58 (1.7%) cutaneous melanomas*
5/22 (23%)
0/13 (0%)
N-terminal
Extracellular domain
TM
JMD
Exons 1–9
Exon 10
Exon 11
TK-1
A8 2 I8 9P 41 : 2 V: % 2%
ND
D 81 D 6H 82 : Y8 0Y 4.1 23 : 2 % D % :2 %
D el K5 554 5 – Y5 0N 559 5 : : W 3N 2% 2% 5 : K5 57R 2% 5 : V5 8N 6.1 5 : % V5 9A 2% 5 : V5 9D 8.2 6 : % N 0D 2% 56 : V5 6D 2% 6 : L5 9G 2% 7 : D 6P 2% el : In 579 34.7 s5 : % 83 2% :2 % R 63 K6 4W 42 : 2 E: % 16 .3 %
CSD, chronic sun-damaged; ND, not determined. *Study did not subclassify melanomas on sun-exposed skin.
TK-2
ND C-terminal
Exons 12–21
Figure 1. Distribution and frequency of published KIT mutations in melanoma. (Went et al., 2004; Willmore-Payne et al., 2005, 2006; Curtin et al., 2006; Antonescu et al., 2007; Beadling et al., 2008; Hodi et al., 2008; Lutzky et al., 2008; Quintas-Cardama et al., 2008; Rivera et al., 2008; Satzger et al., 2008; Ashida et al., 2009). No detailed mutation analyses have been reported on exons 1–8 and exons 19–21; ND, not determined.
melanoma types and further support the notion that these melanoma types are biologically distinct. KIT AS A THERAPEUTIC TARGET The clinical importance of KIT mutations in melanoma relates to the fact that approved drugs are available for inhibition of its kinase activity and are already used successfully in other cancers. The mutation spectrum of KIT seen in melanoma partially overlaps with that seen in gastrointestinal stromal tumor (GIST), where most mutations also occur in exon 11. GIST is the most common mesenchymal tumor of the gastrointestinal tract and has been shown to be particularly sensitive to the receptor tyrosine kinase inhibitors of KIT. Exon 11 encodes the juxtamembrane domain (Figure 1), which has an a-helical conformation and provides a critical auto-inhibitory function, which is disrupted by mutations that change the amino-acid sequence (Wardelmann et al., 2007). The spectrum of exon-11 mutations in melanoma and GIST (65–70%) consists of point mutations as well as in-frame deletions and insertions, which have 22
been shown to promote KIT dimerization in the absence of stem-cell factor and release the receptor from its autoinhibited conformation, resulting in constitutive activation (Debiec-Rychter et al., 2006; Hornick and Fletcher, 2007). GISTs with exon-11 mutations respond well to imatinib, a competitive inhibitor of BCR-ABL, ARG (ABL-related-gene), KIT, PDGFRA, and PDGFRB tyrosine kinases (Heinrich et al., 2003, 2006). Overall, more than 80% of patients with advanced GIST experience an objective clinical benefit showing partial response or stable disease to imatinib therapy (Demetri et al., 2002; Hornick and Fletcher, 2007). The median response duration exceeds 2 years, and patients with GIST diagnosed with specific mutations expect to live for a median of about 5 years compared with only a year in the pre-imatinib era (Guo et al., 2007). However, responses to imatinib depend on the functional domain affected (Heinrich et al., 2003). GIST patients with exon-11 mutations show partial responses in about 61.3–83.5% of cases and 8.2–31.9% show stable disease in response to imatinib
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(Heinrich et al., 2003; Debiec-Rychter et al., 2006). By contrast, mutations in exon 9 of KIT (10–15%), which encodes the fifth extracellular immunoglobulinlike loop, are less responsive, although this limitation can be partially overcome by a high-dose regimen (400 mg twice daily) (Debiec-Rychter et al., 2006). Different from melanoma, KIT mutations located in exon 13 (encoding the tyrosine kinase-1) and exon 17 (encoding the tyrosine kinase-2) occur only in a minority of GISTs (o2 and o1%, respectively) (Debiec-Rychter et al., 2006). Owing to the relative infrequency of GIST, the therapeutic experience with imatinib with these types of mutations is less extensive, although responses have been reported (Hornick and Fletcher, 2007). About 10–20% of GIST patients show primary resistance to imatinib, mostly because of mutations in the receptor that are not covered by imatinib or mutations in other, yet to be discovered, genes (Heinrich et al., 2003; Debiec-Rychter et al., 2006). To date, only anecdotal reports of melanoma patients with KIT mutations and early data from one of the phase-II
MC Garrido and BC Bastian KIT as a Therapeutic Target in Melanoma
trials are available. While still preliminary, these data are so encouraging that the hope that therapeutic successes in GIST may translate to melanomas with KIT alterations seems justified. Two patients with mucosal melanomas, harboring mutations in exons 11 and 13, respectively, have demonstrated dramatic responses to imatinib (Hodi et al., 2008; Lutzky et al., 2008), showing near complete responses. The recently presented preliminary data from the first twelve patients of a phase-II trial of imatinib in melanoma patients with KIT mutations or amplifications showed two complete remissions, two partial remissions, six patients with stable disease, and two with progressive disease (Carvajal R. et al. ASCO 2009, abstract 9001). The importance of proper patient selection is highlighted by the negative experience with imatinib in previous trials with unselected melanoma patients. Three phase-II trials of imatinib in metastatic melanomas were conducted before the discovery of KIT mutations in melanoma and have proven mostly disappointing (Ugurel et al., 2005; Wyman et al., 2006; Kim et al., 2008a). In retrospect, one of the trials found a near-complete response of a patient with metastatic acral melanoma at 12 months of treatment (Kim et al., 2008a), consistent with the finding of genetic alterations of KIT in acral melanomas. This patient, although showing high KIT-receptor expression by immunohistochemistry, did not harbor a detectable mutation, although no amplification study was performed. Together, these results have renewed the enthusiasm for KIT inhibitors and indicate the promise of treatments targeted against specific genetic alterations in melanoma. The results to date are particularly encouraging, as they occur in acral and mucosal melanomas, melanoma categories that are characterized by high degree of chromosomal instability with numerous amplifications (Curtin et al., 2005), and an aggressive disease course that has shown little response to existing treatments. The observation that responses seem to occur despite the presence of numerous other genetic alterations is indicative that melanomas
with KIT mutations are ‘‘addicted’’ to KIT activation. The concept of oncogene addition, first described by Weinstein (2002), posits a critical dependence on an activated oncoprotein. One proposed mechanism includes differential attenuation rates of the pro-survival and proapoptotic signals downstream of many activated kinases. On acute inhibition, the prosurvival signals attenuate faster than the proapoptotic signals, resulting in cell death (Sharma et al., 2006, 2007). Three main pathways appear to be affected by KIT inhibition in melanoma, mitogen-activated protein kinase, and phosphatidylinositol-30 -kinase, STAT, as has been recently shown in cell cultures derived from patient metastases treated with imatinib (Jiang et al., 2008). LESSONS FROM THE USE OF KINASE INHIBITORS IN OTHER CANCER TYPES Although the experience in treating melanoma patients with inhibitors of KIT is just emerging, several lessons can be learned from the use of receptor tyrosine kinase inhibitors in other cancers. Non-small-cell lung cancers have amplifications or activating mutations of the epidermal growth factor receptor (EGFR) in 10–60% of cases, whereas 10–30% have mutations in KRAS (Ahrendt et al., 2001; Eberhard et al., 2005; Bonomi et al., 2007). Both activate the mitogen-activated protein kinase and phosphatidylinositol-30 -kinase/AKT pathways, similar to KIT. The experience in lung cancer indicates that if the pathways are activated at the level of EGFR, targeted agents against the receptor are effective (Eberhard et al., 2005). By contrast, if the pathways are activated at the level of RAS, that is, downstream of the receptor, no responses to anti-EGFR therapy can be expected. Other than in melanoma, in which RAS mutations occur almost exclusively in NRAS, KRAS mutations are found in nonsmall-cell lung cancers. More recent studies suggest that patients with KRAS mutations not only fail to benefit from erlotinib, but may experience decreased survival and ‘‘time to progression’’
(Eberhard et al., 2005; Bonomi et al., 2007). However, the picture is confounded by the fact that cancers with EGFR activation have better prognosis than those with RAS activation independent of treatment (Kim et al., 2008b). Although better prognosis of, for example, KIT-mutant acral or mucosal melanomas compared with similar melanoma types without mutations seems unlikely, this question needs to be addressed in future studies. Interestingly, co-administration of targeted agents with conventional chemotherapy appears to be contra-productive in lung cancers with EGFR mutations and can even defeat the effects of targeted agents (Sharma et al., 2006, 2007). This is because chemotherapeutic agents may promote cell-cycle arrest and thereby suppress apoptosis triggered by acute inactivation of an oncogenic kinase achieved through targeted therapy. This mechanism has potentially contributed to the disappointing results observed when EGFR kinase inhibitors were administered together with conventional chemotherapy drugs in non-small-cell lung cancer (Sharma et al., 2006). Although it is not clear whether the same would apply to melanomas treated with KIT inhibitors, these observations at least raise caution for combination approaches with conventional chemotherapy. BIOMARKERS OF RESPONSE TO KIT INHIBITORS IN MELANOMA The pattern of mutually exclusive mutations in EGFR and KRAS in nonsmall-cell lung cancers is somehow analogous to that in melanomas. In melanomas, KIT, NRAS, BRAF, and GNAQ mutations occur in virtually exclusive patterns, indicating that they provide partially redundant functions. On the basis of these assumptions, it seems unlikely that melanoma patients with BRAF, NRAS, or GNAQ mutations will respond to treatments directed at KIT. The limited data on melanoma to date indicate that the experience in GIST may translate to melanoma: cases with mutations will respond to drugs active against the specific oncoprotein. Although KIT mutations are frequently accompanied by overexpression of KIT www.jidonline.org
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MC Garrido and BC Bastian KIT as a Therapeutic Target in Melanoma
protein, overexpression alone appears not to be sufficiently specific, as clinical trials using expression alone as a marker have not been able to identify responders. In GIST, CD117 expression and KIT genotype do not always correlate (Medeiros et al., 2004). In melanoma, KIT-mutant cases can lack detectable expression of CD117, so that a negative CD117 stain cannot be reliably used to rule out a mutation (Curtin et al., 2006; Beadling et al., 2008; Rivera et al., 2008). Although Curtin et al. (2006) showed KIT expression in most of their initially negative cases when a 10-fold higher concentration of the antibody was used; false-positive staining at such antibody concentration has been reported (Hornick and Fletcher, 2002). Important questions that need to be answered in ongoing and future trials with KIT inhibitors include the nature of indicators of response, mutations, and possibly copy-number increases, which are good candidates, but other biomarkers may emerge. For now, melanomas arising from acral, mucosal, and CSD sites (the head and neck and chronically Sun-exposed extremities of older patients) are to be considered enriched for KIT mutations or amplifications, whereas mutations are expected to be infrequent in the non-CSD melanoma category that affects the not chronically Sun-exposed skin (trunk, extremities) of younger individuals. MONITORING RESPONSE TO THERAPY Although the anecdotal reports of responses of melanomas to KIT inhibitors such as imatinib have been dramatic and easy to detect, more subtle responses may occur in other patients. In GIST, metabolic responses seen on positron emission tomography using fluorine-18-fluorodeoxyglucose (18FDG) can precede significant decrease in tumor size on computed tomography by weeks or months and can be seen as an early-response indicator (Hofman et al., 2007). Conversely, lack of metabolic response on FDG-positron emission tomography indicates primary resistance to the drug, and re-emergence of metabolic activity 24
within tumor sites following a period of therapeutic response indicates secondary resistance to the drug (Van Den Abbeele, 2008). Ongoing clinical trials in melanoma will provide information regarding which methods are likely to assess and monitor responses under therapy with KIT inhibitors. RESISTANCE TO THERAPY About 87% of KIT mutations occur in exons 11 and 13 in melanoma and are, therefore, potentially imatinib-sensitive. However, despite the remarkable initial response to imatinib therapy in several cancers, resistance frequently occurs during treatment. This secondary or acquired resistance has to be distinguished from primary resistance, which can be caused by mutations in portions of the gene that are not inhibited by the drug or by the presence of additional genetic factors. In GIST, acquired resistance to imatinib typically occurs through second-site mutations in KIT, which bypass the inhibitory effects of the drug either by interfering with drug binding or by activating a different portion of the receptor unaffected by the drug. The fact that resistance occurs at the level of KIT and not by additional mutations in downstream components or other signaling pathways is the most stunning illustration of the specificity of oncogene addiction and underscores the unique role of KIT as a therapeutic target in these tumors. In GIST, the second-site mutations occur without exception on the same allele as the primary mutation and affect either the first or the second kinase domains (exons 13, 14, and 17), leading to an imatinib-resistant KIT oncoprotein (Heinrich et al., 2006). In some cases, multiple secondary KIT mutations can be detected in addition to the primary mutation (Heinrich et al., 2006). It is likely that imatinib-resistant, KIT-mutant sub-clones already exist at low levels in untreated GISTs and undergo positive selection during therapy rather than representing de novo arising mutations under therapy (Fletcher and Rubin, 2007). Exposure to imatinib may, thus, drive the selection of double KIT-mutant, imatinib-resistant clones. In GIST, two of the most common
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recurrent secondary mutations are V654A and T670I (Prenen et al., 2006). Therapeutic options for patients with GIST who progress on imatinib include dose escalation or treatment with alternative tyrosine-kinase inhibitors. Sunitinib, an approved drug with proven efficacy against KIT, is specifically effective in GISTs with exon-9 mutations, and also has good inhibitory activity against the V654A and T670I mutations in KIT (Prenen et al., 2006). It is also an orally active drug that inhibits multiple receptor tyrosine kinases, including PDGFRA, vascular endothelial growth factor receptor FLT3 (FMS-like tyrosine kinase-3), and KIT (Sakamoto, 2004). Similar to imatinib, the efficacy of sunitinib in the treatment of imatinib-resistant GISTs depends on the type of mutation (Prenen et al., 2006), and the emergence of resistant clones with second-site mutations has been described as well, primarily in exon 17 (Liegl et al., 2008). Additional KIT inhibitors that are already in clinical use include nilotinib, sorafenib, and dasatinib (Guo et al., 2007). Nilotinib (AMN107), a multi-targeted kinase inhibitor, selectively inhibits both wild-type and KIT mutants in exon 11 (V560del and V560G) and exon 13 (K642E), and is active against KIT double mutants involving exon 11 and exons 13 or 17 (Weisberg et al., 2005; Verstovsek et al., 2006; Guo et al., 2007). By contrast, nilotinib does not appear to be effective against the KIT exon 14 (T670I) mutants. Dasatinib is an ATPcompetitor that inhibits both wild type and KIT with juxtamembrane domain mutations (V559D and V560G) more potently than imatinib (Schittenhelm et al., 2006). It is also active against the double-mutant KIT (V560G/ D816V), as well as exon 13 or 17 single or double mutants, whereas it has no effect on KIT mutant (T670I) oncoprotein (Schittenhelm et al., 2006). Also, sorafenib (BAY 43-9006), initially developed as inhibitor of the serine–threonine kinase RAF, also has activity against KIT as well as vascular endothelial growth factor receptor-2, vascular endothelial growth factor receptor-3, and PDGFRB (Guo et al., 2007). Sorafenib has been shown to
MC Garrido and BC Bastian KIT as a Therapeutic Target in Melanoma
inhibit KIT in in vitro assays (Wilhelm et al., 2004), including the T670I KIT mutation as single or double-mutant Ba/F3 cell lines (Guo et al., 2007), and in vivo the first case showing complete response of a melanoma patient harboring the V560D mutation has recently been reported (QuintasCardama et al., 2008). Concurrent genetic alterations in addition to mutation or amplification of KIT may also modify the tumor’s response to therapy. One alteration that is frequently found in acral and CSD melanomas is amplification of cyclinD1, which acts downstream of KIT at the junction to the cell-cycle entry checkpoint (Sauter et al., 2002, Curtin et al., 2005). Cyclin-D1 is amplified in about 40% of acral melanomas and extra copies are also found in up to 50% of CSD melanomas. On the basis of these observations, we have previously suggested that increased copies of cyclin-D1 may blunt the response to upstream inhibition (Curtin et al., 2005), and this has recently been confirmed for melanoma cells with BRAF mutations (Smalley et al., 2008). CONSIDERATIONS OF WHEN TO INITIATE TREATMENT The phenomenon of secondary resistance because of the presence of second-site mutant cells at the time of therapy initiation strongly argues that treatment should be initiated at low tumor burden to maximize the chances of success. The smaller the number of tumor cells at therapy initiation, the smaller are the chances of the presence of a genetic variant that carries resistant mutations. Another factor supporting treatment at earlier stages is the observation that the central nervous system represents a sanctuary site for certain drugs and that survival prolongation may be limited once metastatic cells have reached the brain. This pattern has also been observed in preclinical mouse models of chronic myeloid leukemia with development of central nervous system involvement under imatinib, whereas systemic disease was fully controlled (Wolff et al., 2003). There have been similar experiences in patients with chronic myeloid leukemia (Petzer et al., 2002) and GIST
(Hughes et al., 2004) where cerebral relapses are observed, whereas systemic disease progression is controlled by imatinib. Other KIT inhibitors such as dasatinib appear to have better penetration of the blood–brain barrier (Porkka et al., 2008), and their efficacy in treating manifest disease in the brain needs to be proven. Even if brain metastases occur under therapy, it may be important to maintain treatment to control disease progression outside of the brain, whereas cerebral metastases are addressed surgically or radiotherapeutically (Hughes et al., 2004). CONCLUSION In summary, the emerging data on KIT inhibitor therapy in melanoma indicate that these drugs will be an effective therapy for patients selected on the basis of their underlying genetic alterations. If confirmed, this would represent a paradigm-shifting entry into an era of personalized medicine for melanoma patients, where therapy can be tailored to mechanistically relevant changes in the cancer cells. Although primary resistance by mechanisms yet to be discovered may make treatment ineffective in some patients, yet this new development raises the hope that melanomas with mutations in other oncogenes may also become treatable once effective drugs against them are found. CONFLICT OF INTEREST The authors state no conflict of interest.
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KIT as a Therapeutic Target in Melanoma Maria C. Garrido1 and Boris C. Bastian1,2 Recently, genetic alterations activating the receptor tyrosine kinase KIT have been shown in some types of melanoma. KIT mutations can be successfully targeted by approved drugs in other cancers. Emerging evidence suggests that melanomas with KIT activation may also respond to KIT inhibitors that are already in clinical use. If confirmed in ongoing clinical trials, this experience would underscore the importance of recognizing the biological diversity among melanomas, representing a first decisive step toward the individualized and mechanism-based treatment of melanoma. Journal of Investigative Dermatology (2010) 130, 20–27; doi:10.1038/jid.2009.334; published online 22 October 2009
INTRODUCTION KIT is a member of the type-III transmembrane receptor tyrosine kinase familythat comprises five extracellular immunoglobulin domains, a single transmembrane region, an inhibitory cytoplasmic juxtamembrane domain, and a split cytoplasmic kinase domain separated by a kinase insert segment (Yarden et al., 1987). Under physiological conditions, binding of the KIT ligand, stem-cell factor, to the extracellular domain of the receptor results in receptor dimerization, activation of the intracellular tyrosine kinase domain through autophosphorylation of specific tyrosine residues, and receptor activation (Lev et al., 1992). The downstream signal transduction pathways include the mitogen-activated protein kinase, phosphatidylinositol-30 -kinase, and JAK/STAT (JAK/signal transducers and activators of transcription) pathways. The intracellular signaling through KIT plays a critical role in the development of several mammalian cell types, including melanocytes, hematopoietic progenitor cells, mast cells, primordial germ cells, and interstitial cells of Cajal (Nishikawa et al., 1991; Galli et al., 1995). Although it has been extensively shown that KIT
signaling is essential for melanocyte development, its precise role in migration, survival, proliferation, and differentiation remains incompletely understood. KIT function is important for survival of melanoblasts, as melanocyte precursors with loss-of-function mutations in KIT never disperse and ultimately disappear (Wehrle-Haller and Weston, 1995). In mouse, survival of melanogenic subpopulations of neural crest cells depends on stem-cell factor for a critical period, which begins after the second day of dispersal, and lasts for about 4 days, ending about the time that melanocytes terminally differentiate, as indicated by the presence of functional melanosomes (Morrison-Graham and Weston, 1993). A role for KIT in the dorsoventral migration of melanocytes is also indicated by the fact that loss-of-function mutations in the KIT pathway lead to patterned pigmentation phenotypes such as white midline spotting in animals and piebaldism in humans (Giebel and Spritz, 1991). Specifically, KIT activation has been shown to be transiently required in the dorsal dermatome before the onset of trunk neural crest dispersal on the lateral pathway (Wehrle-Haller and Weston,
1995). Alexeev and Yoon (2006) have shown that activation of the KIT receptor primarily results in stimulation of migration rather than proliferation of melanocytes and found that it is also responsible for morphological changes of melanocytes such as spindle-shaped bodies and reduced number of dendrites (Alexeev and Yoon, 2006). KIT activation in melanocytes results in rapid increase in actin stress-fiber formation and elevated melanocyte migration on fibronectin (Scott et al., 1996), indicating a role for KIT in the reorganization of the cytoskeleton and higher migratory properties of melanocytes. When melanocytes with activating KIT mutations are transplanted into the dorsal skin of albino mice, cells show a distinctive migration pattern, from the injected sites to the upper dermis and dermal–epidermal border, toward the follicular and/or interfollicular keratinocytes (Kunisada et al., 1998). Despite clear evidence that KIT activation is linked to phenotypes such as migration and survival associated with cancer cells, its role as an oncogene melanoma did not immediately become clear. Early studies of KIT in melanoma lesions found its
1
Department of Dermatology and UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA and 2Department of Pathology and UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA Correspondence: Dr Boris C. Bastian, Departments of Dermatology and Pathology, University of California, San Francisco, UCSF Helen Diller Family Comprehensive Cancer Center, Box 0808, San Francisco, California 94143-0808, USA. E-mail: [email protected] Abbreviations: CSD, chronic sun-damaged; GIST, gastrointestinal stromal tumor; PDGFR, platelet-derived growth factor receptor; PI3K, phosphatidylinositol30 -kinase Received 10 April 2009; revised 28 June 2009; accepted 31 July 2009; published online 22 October 2009
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MC Garrido and BC Bastian KIT as a Therapeutic Target in Melanoma
expression downregulated frequently during progression from early to advanced lesions (Lassam and Bickford, 1992; Natali et al., 1992; Montone et al., 1997), and functional studies even showed that KIT had antiproliferative and antimetastatic properties in some settings (Zakut et al., 1993; Huang et al., 1996). When expressed in melanoma cell lines without KIT expression, KIT induced cell-cycle arrest and apoptosis (Huang et al., 1996). Also, although constitutive activation of KIT in primary melanocytes resulted in increased migration, it also decreased proliferation (Alexeev and Yoon, 2006). These observations have led to the view that KIT may rather act as a tumor suppressor in melanoma, and that to acquire proliferative advantage and escape from the epidermal boundaries, transformed melanocytes need to lose KIT expression. Our attention to KIT as a potential melanoma oncogene was first attracted by a narrow sub-centromeric amplification on chromosome 4q that we observed in acral melanoma, and which appeared to include the KIT locus (Bastian et al., 1998). However, the method of chromosome-based comparative genomic hybridization used at the time did not provide the resolution to sufficiently narrow the affected genomic region, and the published data on the tumor-suppressive effects of KIT at the time appeared to be inconsistent with KIT as a compelling driver gene within the amplicon. Our interest in KIT rose again when we observed a striking field effect in acral melanomas, in which we found single basal melanocytes in the basilar nonlesional epidermis adjacent to acral melanomas that shared similar chromosomal aberrations with the nearby melanoma (Bastian et al., 2000). The extent of these field cells was substantial, reaching up to 2 cm in some cases, suggesting that the melanoma cells were highly migratory, but still under homeostatic control, as they were not increased in number and still equidistantly spaced just as normal melanocytes (North et al., 2008). When BRAF was discovered by the Sanger Center as a melanoma oncogene, we conducted follow-up studies of a larger series of
primary melanomas and found that BRAF mutations were infrequent in mucosal, acral, and chronic sundamaged (CSD) melanomas (Maldonado et al., 2003). The relative dearth of BRAF mutations in these three categories of melanoma, which share a common lentigenous growth pattern characterized by single melanocytes distributed along the basilar epidermis as a common morphological feature, raised the question of what oncogene was activated in these melanoma types, if not BRAF. The lentigenous growth pattern along with the field effect in acral melanoma raised the possibility that a gene inducing melanocyte migration may be involved. A larger study using array comparative genomic hybridization provided us with a refined boundary of the amplicon on chromosome 4q and confirmed that KIT was included in the region, residing at the peak of the amplicon. It was also remarkable that the amplification was virtually exclusive with mutations in BRAF or NRAS (Curtin et al., 2006). As KIT was known to induce melanocyte migration and because of its functional overlap with BRAF and NRAS, KIT rose to the top of the list of candidate genes within the region. When we sequenced KIT, we found recurrent mutations in KIT in mucosal (21%), acral (11%), and CSD melanomas (17%), but not in nonCSD melanomas, in which BRAF mutations predominate. At the time of publication of our results, others had already described occasional mutations of KIT in a few cases of melanomas. Went et al. (2004) performed immunohistochemistry for KIT protein and found expression in 36% (14 of 39). One of the cases with positive expression showed a mutation in exon 11 (L576P). Another study analyzed 100 melanoma metastases not characterized further and showed expression in 29 cases (29%), two of which had mutations, also in exon 11 (L576P) (Willmore-Payne et al., 2005). In addition to KIT mutations, we found frequent copy-number increases of the KIT locus in the same three melanoma categories, which also tended to be mutually exclusive of mutations in BRAF and NRAS. This suggested that copy-number increases of wild-type
KIT may drive these melanomas, or that the copy-number increases at 4q12 implicate another nearby oncogene. The obvious candidate was plateletderived growth factor receptor-A (PDGFRA), a related receptor tyrosine kinase involved in multiple cancer types. However, mutation analysis of PDGFRA in melanomas with and without copy-number increases at 4q12 did not reveal any mutations of PDGFRA. It, therefore, remains plausible that the increased gene dosage of KIT may be pathogenetically relevant in some melanomas. Mutations and copy-number increases in KIT were mostly mutually exclusive. However, mutations at codon K642E were frequently accompanied by amplifications of the mutated allele. This mutation has also been reported in the germ line of patients with familial gastrointestinal stromal tumors, indicating that it may be a weak gain-of-function allele that is tolerable in the germ line (Isozaki et al., 2000), but needs to be increased in gene dosage by amplification to be fully oncogenic. A recent, larger follow-up study has confirmed the recurrent mutations in acral and mucosal melanomas, but did not distinguish between CSD and non-CSD melanomas (Beadling et al., 2008). Table 1 summarizes the mutations reported in KIT to date and their relative distribution across melanoma subtypes. We would like to note that the finding of known oncogenic mutations in KIT in melanoma subsets is not necessarily contradictory to the findings of the tumor-suppressive effects of KIT reported earlier. In fact, the discrepancies between our results and previous reports in the literature (Lassam and Bickford, 1992; Natali et al., 1992; Montone et al., 1997) could be reconciled by the fact that those studies were conducted on melanoma cell lines, which are usually derived from the melanoma subtype characterized by its occurrence on intermittently exposed skin (non-CSD). Together with the dearth of KIT mutations or copy-number increases of KIT in non-CSD melanoma, the divergent findings may, therefore, indicate different roles of KIT signaling in these www.jidonline.org
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MC Garrido and BC Bastian KIT as a Therapeutic Target in Melanoma
Table 1. KIT mutations in melanomas subtypes Reference
Mucosal melanomas
Acral melanomas
CSD melanomas
0/6 (0%)
2/22 (9%)
ND
ND
ND
Ashida et al. (2009) Rivera et al. (2008)
4/18 (22.2%)
Curtin et al. (2006)
8/38 (21%)
3/28 (11%)
3/18 (16.7%)
Satzger et al. (2008)
6/37 (16%)
ND
ND
Antonescu et al. (2007)
3/20 (15%)
Beadling et al. (2008)
7/45 (15.6%)
Carvajal et al. (2009)
12/45 (27%)
ND
ND
3/13 (23%)
1/58 (1.7%) cutaneous melanomas*
5/22 (23%)
0/13 (0%)
N-terminal
Extracellular domain
TM
JMD
Exons 1–9
Exon 10
Exon 11
TK-1
A8 2 I8 9P 41 : 2 V: % 2%
ND
D 81 D 6H 82 : Y8 0Y 4.1 23 : 2 % D % :2 %
D el K5 554 5 – Y5 0N 559 5 : : W 3N 2% 2% 5 : K5 57R 2% 5 : V5 8N 6.1 5 : % V5 9A 2% 5 : V5 9D 8.2 6 : % N 0D 2% 56 : V5 6D 2% 6 : L5 9G 2% 7 : D 6P 2% el : In 579 34.7 s5 : % 83 2% :2 % R 63 K6 4W 42 : 2 E: % 16 .3 %
CSD, chronic sun-damaged; ND, not determined. *Study did not subclassify melanomas on sun-exposed skin.
TK-2
ND C-terminal
Exons 12–21
Figure 1. Distribution and frequency of published KIT mutations in melanoma. (Went et al., 2004; Willmore-Payne et al., 2005, 2006; Curtin et al., 2006; Antonescu et al., 2007; Beadling et al., 2008; Hodi et al., 2008; Lutzky et al., 2008; Quintas-Cardama et al., 2008; Rivera et al., 2008; Satzger et al., 2008; Ashida et al., 2009). No detailed mutation analyses have been reported on exons 1–8 and exons 19–21; ND, not determined.
melanoma types and further support the notion that these melanoma types are biologically distinct. KIT AS A THERAPEUTIC TARGET The clinical importance of KIT mutations in melanoma relates to the fact that approved drugs are available for inhibition of its kinase activity and are already used successfully in other cancers. The mutation spectrum of KIT seen in melanoma partially overlaps with that seen in gastrointestinal stromal tumor (GIST), where most mutations also occur in exon 11. GIST is the most common mesenchymal tumor of the gastrointestinal tract and has been shown to be particularly sensitive to the receptor tyrosine kinase inhibitors of KIT. Exon 11 encodes the juxtamembrane domain (Figure 1), which has an a-helical conformation and provides a critical auto-inhibitory function, which is disrupted by mutations that change the amino-acid sequence (Wardelmann et al., 2007). The spectrum of exon-11 mutations in melanoma and GIST (65–70%) consists of point mutations as well as in-frame deletions and insertions, which have 22
been shown to promote KIT dimerization in the absence of stem-cell factor and release the receptor from its autoinhibited conformation, resulting in constitutive activation (Debiec-Rychter et al., 2006; Hornick and Fletcher, 2007). GISTs with exon-11 mutations respond well to imatinib, a competitive inhibitor of BCR-ABL, ARG (ABL-related-gene), KIT, PDGFRA, and PDGFRB tyrosine kinases (Heinrich et al., 2003, 2006). Overall, more than 80% of patients with advanced GIST experience an objective clinical benefit showing partial response or stable disease to imatinib therapy (Demetri et al., 2002; Hornick and Fletcher, 2007). The median response duration exceeds 2 years, and patients with GIST diagnosed with specific mutations expect to live for a median of about 5 years compared with only a year in the pre-imatinib era (Guo et al., 2007). However, responses to imatinib depend on the functional domain affected (Heinrich et al., 2003). GIST patients with exon-11 mutations show partial responses in about 61.3–83.5% of cases and 8.2–31.9% show stable disease in response to imatinib
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(Heinrich et al., 2003; Debiec-Rychter et al., 2006). By contrast, mutations in exon 9 of KIT (10–15%), which encodes the fifth extracellular immunoglobulinlike loop, are less responsive, although this limitation can be partially overcome by a high-dose regimen (400 mg twice daily) (Debiec-Rychter et al., 2006). Different from melanoma, KIT mutations located in exon 13 (encoding the tyrosine kinase-1) and exon 17 (encoding the tyrosine kinase-2) occur only in a minority of GISTs (o2 and o1%, respectively) (Debiec-Rychter et al., 2006). Owing to the relative infrequency of GIST, the therapeutic experience with imatinib with these types of mutations is less extensive, although responses have been reported (Hornick and Fletcher, 2007). About 10–20% of GIST patients show primary resistance to imatinib, mostly because of mutations in the receptor that are not covered by imatinib or mutations in other, yet to be discovered, genes (Heinrich et al., 2003; Debiec-Rychter et al., 2006). To date, only anecdotal reports of melanoma patients with KIT mutations and early data from one of the phase-II
MC Garrido and BC Bastian KIT as a Therapeutic Target in Melanoma
trials are available. While still preliminary, these data are so encouraging that the hope that therapeutic successes in GIST may translate to melanomas with KIT alterations seems justified. Two patients with mucosal melanomas, harboring mutations in exons 11 and 13, respectively, have demonstrated dramatic responses to imatinib (Hodi et al., 2008; Lutzky et al., 2008), showing near complete responses. The recently presented preliminary data from the first twelve patients of a phase-II trial of imatinib in melanoma patients with KIT mutations or amplifications showed two complete remissions, two partial remissions, six patients with stable disease, and two with progressive disease (Carvajal R. et al. ASCO 2009, abstract 9001). The importance of proper patient selection is highlighted by the negative experience with imatinib in previous trials with unselected melanoma patients. Three phase-II trials of imatinib in metastatic melanomas were conducted before the discovery of KIT mutations in melanoma and have proven mostly disappointing (Ugurel et al., 2005; Wyman et al., 2006; Kim et al., 2008a). In retrospect, one of the trials found a near-complete response of a patient with metastatic acral melanoma at 12 months of treatment (Kim et al., 2008a), consistent with the finding of genetic alterations of KIT in acral melanomas. This patient, although showing high KIT-receptor expression by immunohistochemistry, did not harbor a detectable mutation, although no amplification study was performed. Together, these results have renewed the enthusiasm for KIT inhibitors and indicate the promise of treatments targeted against specific genetic alterations in melanoma. The results to date are particularly encouraging, as they occur in acral and mucosal melanomas, melanoma categories that are characterized by high degree of chromosomal instability with numerous amplifications (Curtin et al., 2005), and an aggressive disease course that has shown little response to existing treatments. The observation that responses seem to occur despite the presence of numerous other genetic alterations is indicative that melanomas
with KIT mutations are ‘‘addicted’’ to KIT activation. The concept of oncogene addition, first described by Weinstein (2002), posits a critical dependence on an activated oncoprotein. One proposed mechanism includes differential attenuation rates of the pro-survival and proapoptotic signals downstream of many activated kinases. On acute inhibition, the prosurvival signals attenuate faster than the proapoptotic signals, resulting in cell death (Sharma et al., 2006, 2007). Three main pathways appear to be affected by KIT inhibition in melanoma, mitogen-activated protein kinase, and phosphatidylinositol-30 -kinase, STAT, as has been recently shown in cell cultures derived from patient metastases treated with imatinib (Jiang et al., 2008). LESSONS FROM THE USE OF KINASE INHIBITORS IN OTHER CANCER TYPES Although the experience in treating melanoma patients with inhibitors of KIT is just emerging, several lessons can be learned from the use of receptor tyrosine kinase inhibitors in other cancers. Non-small-cell lung cancers have amplifications or activating mutations of the epidermal growth factor receptor (EGFR) in 10–60% of cases, whereas 10–30% have mutations in KRAS (Ahrendt et al., 2001; Eberhard et al., 2005; Bonomi et al., 2007). Both activate the mitogen-activated protein kinase and phosphatidylinositol-30 -kinase/AKT pathways, similar to KIT. The experience in lung cancer indicates that if the pathways are activated at the level of EGFR, targeted agents against the receptor are effective (Eberhard et al., 2005). By contrast, if the pathways are activated at the level of RAS, that is, downstream of the receptor, no responses to anti-EGFR therapy can be expected. Other than in melanoma, in which RAS mutations occur almost exclusively in NRAS, KRAS mutations are found in nonsmall-cell lung cancers. More recent studies suggest that patients with KRAS mutations not only fail to benefit from erlotinib, but may experience decreased survival and ‘‘time to progression’’
(Eberhard et al., 2005; Bonomi et al., 2007). However, the picture is confounded by the fact that cancers with EGFR activation have better prognosis than those with RAS activation independent of treatment (Kim et al., 2008b). Although better prognosis of, for example, KIT-mutant acral or mucosal melanomas compared with similar melanoma types without mutations seems unlikely, this question needs to be addressed in future studies. Interestingly, co-administration of targeted agents with conventional chemotherapy appears to be contra-productive in lung cancers with EGFR mutations and can even defeat the effects of targeted agents (Sharma et al., 2006, 2007). This is because chemotherapeutic agents may promote cell-cycle arrest and thereby suppress apoptosis triggered by acute inactivation of an oncogenic kinase achieved through targeted therapy. This mechanism has potentially contributed to the disappointing results observed when EGFR kinase inhibitors were administered together with conventional chemotherapy drugs in non-small-cell lung cancer (Sharma et al., 2006). Although it is not clear whether the same would apply to melanomas treated with KIT inhibitors, these observations at least raise caution for combination approaches with conventional chemotherapy. BIOMARKERS OF RESPONSE TO KIT INHIBITORS IN MELANOMA The pattern of mutually exclusive mutations in EGFR and KRAS in nonsmall-cell lung cancers is somehow analogous to that in melanomas. In melanomas, KIT, NRAS, BRAF, and GNAQ mutations occur in virtually exclusive patterns, indicating that they provide partially redundant functions. On the basis of these assumptions, it seems unlikely that melanoma patients with BRAF, NRAS, or GNAQ mutations will respond to treatments directed at KIT. The limited data on melanoma to date indicate that the experience in GIST may translate to melanoma: cases with mutations will respond to drugs active against the specific oncoprotein. Although KIT mutations are frequently accompanied by overexpression of KIT www.jidonline.org
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MC Garrido and BC Bastian KIT as a Therapeutic Target in Melanoma
protein, overexpression alone appears not to be sufficiently specific, as clinical trials using expression alone as a marker have not been able to identify responders. In GIST, CD117 expression and KIT genotype do not always correlate (Medeiros et al., 2004). In melanoma, KIT-mutant cases can lack detectable expression of CD117, so that a negative CD117 stain cannot be reliably used to rule out a mutation (Curtin et al., 2006; Beadling et al., 2008; Rivera et al., 2008). Although Curtin et al. (2006) showed KIT expression in most of their initially negative cases when a 10-fold higher concentration of the antibody was used; false-positive staining at such antibody concentration has been reported (Hornick and Fletcher, 2002). Important questions that need to be answered in ongoing and future trials with KIT inhibitors include the nature of indicators of response, mutations, and possibly copy-number increases, which are good candidates, but other biomarkers may emerge. For now, melanomas arising from acral, mucosal, and CSD sites (the head and neck and chronically Sun-exposed extremities of older patients) are to be considered enriched for KIT mutations or amplifications, whereas mutations are expected to be infrequent in the non-CSD melanoma category that affects the not chronically Sun-exposed skin (trunk, extremities) of younger individuals. MONITORING RESPONSE TO THERAPY Although the anecdotal reports of responses of melanomas to KIT inhibitors such as imatinib have been dramatic and easy to detect, more subtle responses may occur in other patients. In GIST, metabolic responses seen on positron emission tomography using fluorine-18-fluorodeoxyglucose (18FDG) can precede significant decrease in tumor size on computed tomography by weeks or months and can be seen as an early-response indicator (Hofman et al., 2007). Conversely, lack of metabolic response on FDG-positron emission tomography indicates primary resistance to the drug, and re-emergence of metabolic activity 24
within tumor sites following a period of therapeutic response indicates secondary resistance to the drug (Van Den Abbeele, 2008). Ongoing clinical trials in melanoma will provide information regarding which methods are likely to assess and monitor responses under therapy with KIT inhibitors. RESISTANCE TO THERAPY About 87% of KIT mutations occur in exons 11 and 13 in melanoma and are, therefore, potentially imatinib-sensitive. However, despite the remarkable initial response to imatinib therapy in several cancers, resistance frequently occurs during treatment. This secondary or acquired resistance has to be distinguished from primary resistance, which can be caused by mutations in portions of the gene that are not inhibited by the drug or by the presence of additional genetic factors. In GIST, acquired resistance to imatinib typically occurs through second-site mutations in KIT, which bypass the inhibitory effects of the drug either by interfering with drug binding or by activating a different portion of the receptor unaffected by the drug. The fact that resistance occurs at the level of KIT and not by additional mutations in downstream components or other signaling pathways is the most stunning illustration of the specificity of oncogene addiction and underscores the unique role of KIT as a therapeutic target in these tumors. In GIST, the second-site mutations occur without exception on the same allele as the primary mutation and affect either the first or the second kinase domains (exons 13, 14, and 17), leading to an imatinib-resistant KIT oncoprotein (Heinrich et al., 2006). In some cases, multiple secondary KIT mutations can be detected in addition to the primary mutation (Heinrich et al., 2006). It is likely that imatinib-resistant, KIT-mutant sub-clones already exist at low levels in untreated GISTs and undergo positive selection during therapy rather than representing de novo arising mutations under therapy (Fletcher and Rubin, 2007). Exposure to imatinib may, thus, drive the selection of double KIT-mutant, imatinib-resistant clones. In GIST, two of the most common
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recurrent secondary mutations are V654A and T670I (Prenen et al., 2006). Therapeutic options for patients with GIST who progress on imatinib include dose escalation or treatment with alternative tyrosine-kinase inhibitors. Sunitinib, an approved drug with proven efficacy against KIT, is specifically effective in GISTs with exon-9 mutations, and also has good inhibitory activity against the V654A and T670I mutations in KIT (Prenen et al., 2006). It is also an orally active drug that inhibits multiple receptor tyrosine kinases, including PDGFRA, vascular endothelial growth factor receptor FLT3 (FMS-like tyrosine kinase-3), and KIT (Sakamoto, 2004). Similar to imatinib, the efficacy of sunitinib in the treatment of imatinib-resistant GISTs depends on the type of mutation (Prenen et al., 2006), and the emergence of resistant clones with second-site mutations has been described as well, primarily in exon 17 (Liegl et al., 2008). Additional KIT inhibitors that are already in clinical use include nilotinib, sorafenib, and dasatinib (Guo et al., 2007). Nilotinib (AMN107), a multi-targeted kinase inhibitor, selectively inhibits both wild-type and KIT mutants in exon 11 (V560del and V560G) and exon 13 (K642E), and is active against KIT double mutants involving exon 11 and exons 13 or 17 (Weisberg et al., 2005; Verstovsek et al., 2006; Guo et al., 2007). By contrast, nilotinib does not appear to be effective against the KIT exon 14 (T670I) mutants. Dasatinib is an ATPcompetitor that inhibits both wild type and KIT with juxtamembrane domain mutations (V559D and V560G) more potently than imatinib (Schittenhelm et al., 2006). It is also active against the double-mutant KIT (V560G/ D816V), as well as exon 13 or 17 single or double mutants, whereas it has no effect on KIT mutant (T670I) oncoprotein (Schittenhelm et al., 2006). Also, sorafenib (BAY 43-9006), initially developed as inhibitor of the serine–threonine kinase RAF, also has activity against KIT as well as vascular endothelial growth factor receptor-2, vascular endothelial growth factor receptor-3, and PDGFRB (Guo et al., 2007). Sorafenib has been shown to
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inhibit KIT in in vitro assays (Wilhelm et al., 2004), including the T670I KIT mutation as single or double-mutant Ba/F3 cell lines (Guo et al., 2007), and in vivo the first case showing complete response of a melanoma patient harboring the V560D mutation has recently been reported (QuintasCardama et al., 2008). Concurrent genetic alterations in addition to mutation or amplification of KIT may also modify the tumor’s response to therapy. One alteration that is frequently found in acral and CSD melanomas is amplification of cyclinD1, which acts downstream of KIT at the junction to the cell-cycle entry checkpoint (Sauter et al., 2002, Curtin et al., 2005). Cyclin-D1 is amplified in about 40% of acral melanomas and extra copies are also found in up to 50% of CSD melanomas. On the basis of these observations, we have previously suggested that increased copies of cyclin-D1 may blunt the response to upstream inhibition (Curtin et al., 2005), and this has recently been confirmed for melanoma cells with BRAF mutations (Smalley et al., 2008). CONSIDERATIONS OF WHEN TO INITIATE TREATMENT The phenomenon of secondary resistance because of the presence of second-site mutant cells at the time of therapy initiation strongly argues that treatment should be initiated at low tumor burden to maximize the chances of success. The smaller the number of tumor cells at therapy initiation, the smaller are the chances of the presence of a genetic variant that carries resistant mutations. Another factor supporting treatment at earlier stages is the observation that the central nervous system represents a sanctuary site for certain drugs and that survival prolongation may be limited once metastatic cells have reached the brain. This pattern has also been observed in preclinical mouse models of chronic myeloid leukemia with development of central nervous system involvement under imatinib, whereas systemic disease was fully controlled (Wolff et al., 2003). There have been similar experiences in patients with chronic myeloid leukemia (Petzer et al., 2002) and GIST
(Hughes et al., 2004) where cerebral relapses are observed, whereas systemic disease progression is controlled by imatinib. Other KIT inhibitors such as dasatinib appear to have better penetration of the blood–brain barrier (Porkka et al., 2008), and their efficacy in treating manifest disease in the brain needs to be proven. Even if brain metastases occur under therapy, it may be important to maintain treatment to control disease progression outside of the brain, whereas cerebral metastases are addressed surgically or radiotherapeutically (Hughes et al., 2004). CONCLUSION In summary, the emerging data on KIT inhibitor therapy in melanoma indicate that these drugs will be an effective therapy for patients selected on the basis of their underlying genetic alterations. If confirmed, this would represent a paradigm-shifting entry into an era of personalized medicine for melanoma patients, where therapy can be tailored to mechanistically relevant changes in the cancer cells. Although primary resistance by mechanisms yet to be discovered may make treatment ineffective in some patients, yet this new development raises the hope that melanomas with mutations in other oncogenes may also become treatable once effective drugs against them are found. CONFLICT OF INTEREST The authors state no conflict of interest.
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