c-Kit related-diseases and malignancies

KitL/c-Kit are essential in hematopoiesis, gametogenesis, melanogenesis and mast cell physiology and function. Dysregulation of the KitL/c-Kit axis, in most cases, leads to abnormal signaling. C-Kit activating mutations often generate these mechanisms. There are also cases of overexpression of c-Kit and KitL, which leads to the formation of paracrine or autocrine loops. The Loss-Of-Function (LOF) and the Gain-Of-Function (GOF) mutations lead to an alteration of the oncoprotein that modifies its activity. These types of mutations are very common in various types of cancers, such as leukemia, gastrointestinal stromal tumors (GISTs), melanomas, and seminomas.

The c-Kit receptor is the first known example of a spontaneous germline LOF mutation in mice.

Many independent mutations have been described on the W and Sl loci. Mice with variations

on the W or Sl loci have similar phenotypic characteristics (Galli et al., 1994). Mutations in the W locus affect the number and function of hematopoietic stem cells, mast cells, germ cells, and melanoblasts (intrinsic action). Mutations in the Sl locus alter the microenvironment that surrounds these different cell lines (extrinsic action) (Zsebo et al., 1990). As described in Table 2, mutations at the murine W locus result in a LOF effect that elicits an array of developmental defects including anemia, coat color defects, sterility (Russell, 1979), and ICC and mast cells depletion (Kitamura et al., 1978) among others.

Table 2. Phenotype of some W mice. Adapted from (Nocka, Tan, et al., 1990)

Genotype Viability Hematopoiesis Gametogenesis Pigmentation Molecular W/W

viable No anemia fertile spotting

Big deletion

In general, homozygous mutations on the W and Sl loci (W/W or Sl/Sl) alleles produce animals with macrocytic and hypoplastic anemia, with a lack of skin pigmentation (white animals with black eyes), sterile and with a profound deficit in mast cells. These mutations are often dominant negative (McCulloch & Minden, 1993). The most severe homozygous mutants die in utero or the perinatal period due to severe anemia. The Sl mutation has a more pronounced effect and causes a more severe anemia (Galli et al., 1994).

Commonly, single heterozygous mice (W/+) are fertile and have no hematopoiesis failure, their coat is not diluted, but they present a depigmented spot on the belly the forehead and or on the ends of the paws and tail. Heterozygous (Sl/+) mice are viable, they have moderate

macrocytic anemia, a dilated dress with a ventral spot and reduced size but remain fertile (Galli et al., 1994). In mice, it exists two major kinds of mutations:

i) Mutations in the TKD: W⁴², W³⁷, W^v, W⁴¹;

ii) Truncated c-Kit receptor: This include the W variant which carry a deletion of 78 amino acids in the TMD and the C-term of the TKD. It results in a smaller gene and a lack of membrane expression. The W^19H mutation which consists of a complete deletion of the c-Kit gene that causes a total absence of the c-Kit receptor on the cell surface.

The defects in heterozygous mice with the TKD mutations are more severe than those of heterozygous mice that have truncated receptors. These mutations are associated with structural alterations of the c-Kit gene. Some mutations are single base pair substitutions in the kinase domain that result in a partial or complete loss of kinase activity of the c-Kit receptor. In heterozygous states, the more severe the mutation, the higher the loss of function of the Kit receptor and the more the vital role is hindered. Animals that lack any c-Kit tyrosine kinase activity die in utero while those that have a reduced level of kinase activity can survive (McCulloch & Minden, 1993).

V.3.i. Piebaldism

In humans, c-Kit LOF mutation is responsible for an autosomal-dominant disorder known as Piebaldism (Giebel & Spritz, 1991). This syndrome is characterized by congenital non-pigmented patches on the hair and skin, mainly located on the forehead, chest, abdomen, and limbs. Besides the pigmentation defects, patients present mast cell depletion, macrocytic anemia, reduced fertility and in some cases learning impairment (Larizza & Beghini, 2000).

The white patches are present from birth and, in general, do not evolve over the course of life (Lopez & Jorda, 2011). Piebaldism is linked to point mutations (substitutions) in the kinase domain at codon E583, F584, G664 (Galli (Galli et al., 1994) et al., 1994). Because the role of c-Kit in migration, proliferation, differentiation, and survival of melanoblasts, skin lesions are linked to a lack of melanocytes in the affected areas.

The loss of c-Kit expression has also been associated with thyroid cancer, melanoma and breast cancer in humans (Rönnstrand, 2004).

Activating (GOF) mutations of c-Kit have been implicated in tumorigenesis (Figure 9). In humans, these mutations are involved in systemic mastocytosis (Buttner et al., 1998; Nagata et al., 1995), mast cell leukemia (Ning et al., 2001a), acute myeloid leukemia (Ashman et al.,

1999), and sinonasal natural killer/T cell lymphomas (Hongyo et al., 2000). They have also been identified in GISTs (Heinrich et al., 2003; Hirota et al., 1998; Rammohan et al., 2013), germ cell tumors (Rapley et al., 2004), melanoma (Willmore-Payne (Willmore-Payne et al., 2005), and small cell lung cancer (Boldrini et al., 2004).

Figure 9. Distribution of c-Kit mutations in human malignancies. In the center, a schematic representation of the structural domains of c-Kit: The extracellular domain (ECD), the transmembrane domain (TMD), the juxtamembrane domain (JMD), the tyrosine kinase domains (TKD) and the c-tail. The four boxes mention the mutation and the related diseases that have been described for each region.

V.3.ii. Leukemias

Leukemias are a group of pathologies linked to an abnormal accumulation of hematopoietic cells in the bone marrow or the blood. Chronic Myeloid Leukemia (CML) is related to the abnormal proliferation of mature granulocytes. Acute Myeloid Leukemias (AML) is a rapidly developing disease in precursors of undifferentiated hematopoietic cells (blasts) invade the bone marrow and blood. The c-Kit expression is observed in AML (68%) and CML (80%), but

rarely in lymphoid leukemia (Liang et al., 2013) and is correlated with a poor prognosis (Tsao et al., 2004).

Compare to AML, c-Kit mutations are rare events in Core Binding Factor (CBF)-AML (Ashman

& Griffith, 2013). The CBF-AML is an AML sub-type carrying a chromosomal translocation (8;

21) or inversion (inv16) that lead to the fusion proteins AML-ETO and CBF-MYH11 (Higuchi et al., 2002). In these cases, c-Kit mutations are a secondary but crucial event that induces proliferation and survival of leukemia cells (Y. Y. Wang et al., 2011). Approximately 37% of adults and 19% of children affected with CBF-AML have c-Kit activating mutations (Ashman &

Griffith, 2013). Although patients with CBF-AML have a better prognosis, patients with inv(16) have higher rates of relapse if c-Kit is mutated in exon 8 (Care et al., 2003). Mutations in exon 17 are associated with poor prognosis (Liang et al., 2013).

In 2005, Kohl et al. showed that ectopically expression of these mutations in murine cells supports cell proliferation and resistance to apoptosis. In this system, inhibition of PI3K or Mek1 with LY294002 or PD98059, respectively blocked c-Kit-induced cell proliferation (Kohl et al., 2005). These data suggest that such a mutation have a pro-proliferative effect, due to the activation of signaling pathways downstream of the oncogenic receptor.

V.3.iii. Gastrointestinal Stromal Tumors (GISTs)

GISTs are solid mesenchymal tumors located at the level of the digestive tract and extending to the abdomen with the possibility of hepatic dissemination. The disease is characterized by an abnormal proliferation of ICCs. Mutations on c-Kit are the most frequent and accounts for 75-80% of cases (Corless et al., 2011). Such mutations were associated with a constitutively active receptor and support cell proliferation, in the absence of ligand. Also, nude mice grafted with c-Kit activating mutations showed increased tumor growth compared with control mice (Hirota et al., 1998).

Among patients with GIST, c-Kit mutations are heterogeneous. Indeed, 68% of the patients have a mutation in exon 11 (JMD), 10% in exon 9 (ECD) and 1% in exons 13 or 17 (Corless &

Heinrich, 2008). The last mutations are secondary, although rare in nontreated patients, are very frequent after treatment with imatinib and seems to be responsible for resistance (T.

Guo et al., 2007). The c-Kit^V560G mutant results in the constitutive opening of the catalytic pocket (Mol et al., 2003). In 2004, Duensing et al. analyzed the downstream signaling of c-Kit^V560G in primary cells from patients with GIST, as well as on cell lines with activating

mutations of the receptor. This study showed that Erk and Akt are activated in a c-Kit-dependent manner, and this was associated with the pro-proliferative capacity of tumor cells (Duensing et al., 2004). However, in a hematopoietic cell model expressing c-Kit^V560G none of these proteins (Akt and Erk) were phosphorylated (Frost et al., 2002). Therefore, signaling downstream of the same active mutant receptor likely depends on the cellular context.

Finally, not all GIST patients have c-Kit mutations. Activating mutations in the PDGFRa account for 5-8% of GISTs cases. It should be noted that the activating mutations of c-Kit and PDGFRα are mutually exclusive (Corless et al., 2011).

V.3.iv. Systemic mastocytosis

Mastocytosis forms a heterogeneous group of diseases characterized by an abnormal proliferation and accumulation of mast cells in various tissues or organs (Valent et al., 2012).

We can distinguish pediatric mastocytosis (mostly benign), and systemic mastocytosis of the adult which is malignant and aggressive. From biopsies of patients with adult mastocytosis, Longley et al. identified c-Kit activating mutations located in the kinase domain of the receptor (B. J. Longley, Jr. et al., 1999). In most cases, it consists on a substitution of aspartate by valine in position 816, c-Kit^D816V (Lim et al., 2009; Valent, 2008), 2008, Lim et al., 2009). The c-Kit^D816V mutation has been found in more than 80% of cases with systemic mastocytosis (Furitsu et al., 1993; Nagata et al., 1995). There are also other mutations affecting this residue, D816Y, and D816F. All of them confer on the receptor a constitutively active kinase activity, via the permanent alteration of its conformation.

In 2001, Chian et al. showed that PI3K is constitutively activated downstream of c-Kit^D816 mutants (Chian et al., 2001). Besides, c-Kit^D816V activates Akt on primary cells of patients with mastocytosis (Gabillot-Carre et al., 2006). The anti-proliferative effects of specific inhibitors of PI3K (wortmannin or LY249002) indicated that the proliferative capacity of c-Kit^D816V expressing cells depends on PI3K signaling. FES kinases have also been linked to the proliferation of cells from mice carrying the D814Y mutation (human D816V) (Voisset et al., 2007). Mutations affecting the D816 residue increased the activation of STAT3 leading to spontaneous cell proliferation (Ning et al., 2001a, 2001b). In the context of D816, STAT5 is also activated and localized in the cytoplasm where it interacts with Gab2 and PI3K. It has been suggested that c-Kit^D816 induce and oncogenic signaling axis involving STAT5, PI3K, and Akt that supports tumor cell proliferation (Baumgartner et al., 2009; Harir et al., 2008; Harir

et al., 2007). However, it seems that the D816V mutation confers a moderate oncogenic potential. A recent study exposed a negative regulation of this mutant by Y568/570. Indeed, mutation by phenylalanine reinforced the capacity c-Kit^D816V to induce cell growth in semi-solid medium and leukemia when the cells are injected into nude mice (Chaix et al., 2014).

The activating mutation c-Kit^D816V mutation is resistant to imatinib (Akin et al., 2003; Ma et al., 2002; Zermati et al., 2003). It has been proposed that c-Kit^D816V may prevent binding of imatinib by modifying the conformation of the A-loop (Mol et al., 2003). Other drugs such as Dasatinib and Midostaurin are more efficient than Imatinib in inhibiting the kinase activity of c-Kit^D816V (Shah et al., 2006)

V.3.v. Melanomas

In recent years, the incidence of melanoma has increased considerably. The genes often involved are BRAF, NRAS, as well as genes encoding proteins that function in the cell cycle.

Mutations in the BRAF and NRAS genes account for 60% and 10-20% of melanomas, respectively (Curtin et al., 2006). Because of its role in the development of melanocytes, c-Kit has been implicated in some types of melanoma (Beadling et al., 2008). Indeed, acral, mucosal and sun-induced melanomas present c-Kit amplifications and mutations (exons 11, 13, 17, 18) (L. S. Liu & Colegio, 2013). However, the oncogenic role of c-Kit in melanomas still unclear as loss of c-Kit expression is associated with the progression of certain melanoma forms (Lennartsson et al., 2005).

The involvement of c-Kit in melanomas prompted clinical trials with c-Kit targeting drugs including Imatinib (J. Guo et al., 2011), Dasatinib (Kluger et al., 2011) and Nilotinib (Cho et al., 2012). The sensitivity to these inhibitors depends on the mutation type (Carvajal et al., 2011) and will be discussed in section VI.

V.3.vi. Germ cell tumors

Tumors of germ cells, dysgerminomas in women and seminomas in men, express c-Kit (Hoei-Hansen et al., 2007). Given the function of c-Kit in the proliferation and migration of progenitors of germ cells, researchers are interested in its involvement in germ cell tumors.

Indeed, mutations in exon 17, as well as duplications of c-Kit, have been identified in seminomas (Kemmer et al., 2004; McIntyre et al., 2005). Seminomas have a good prognosis with high prognosis rates, where surgical resection is the primary treatment (Oldenburg et

al., 2013). Dysgerminomas, on the other hand, respond well to chemotherapy and radiotherapy and have a good prognosis (Sever et al., 2005).

V.3.vii. Small Cell Lung Cancer (SCLC) and neuroblastoma

The KitL/c-Kit axis form paracrine or autocrine loops that might lead to cancer including SCLC and neuroblastoma.

SCLC is an aggressive tumor with a poor prognosis that represents 20% of lung cancers. It responds to chemotherapy and radiotherapy but systematically relapses. In about 40-80% of cases, c-Kit is expressed and constitutes a factor of poor prognosis. The proliferation of tumor cells results in KitL/c-Kit paracrine or autocrine loops (50%) and much less frequent by c-Kit mutations in exons 9 and 11 (Boldrini et al., 2004; Hibi et al., 1991). The effect of imatinib on SCLC patients expressing c-Kit was assessed in phase II clinical trials without satisfactory results (Dy et al., 2005).

Neuroblastoma is a pediatric tumor that derives from neural crests, from which melanocytes arise. A KitL/c-Kit autocrine loop appears to be essential for tumor proliferation (Cohen et al., 1994). The growth of neuroblastoma cells was shown to be blocked by imatinib in vitro and in vivo by inhibiting the phosphorylation of c-Kit and PDGFR (Beppu et al., 2004). A clinical trial with imatinib has shown encouraging results and good tolerance in neuroblastoma patients (Calafiore et al., 2013).

Lastly, overexpression of c-Kit and the formation of an autocrine KitL/c-Kit loop have been reported in ovarian (Chau et al., 2013), breast (Tsuda et al., 2005), Ewing sarcoma (Gonzalez et al., 2004), and colorectal cancers (Bellone et al., 1997).

The KitL/c-Kit pathway is involved in the physiology of several cell lineages during the embryonic development and adulthood. Given their function in cell proliferation, KitL and its receptor c-Kit are at the origin of the different type of malignancies. Most c-Kit affecting mutations lead to a GOF effect that renders the receptor constitutively active. That is why the receptor c-Kit constitutes a therapeutic target for the development of drugs to treat cancer.