We recently published that c-Kit can be mechanically activated by an anchored-ligand, and that this response is insensitive to imatinib (S. Tabone-Eglinger et al., 2014). We decided to exploit this aspect and developed a functional screening to analyze different TKIs profiles aiming to test various drugs and select those that inhibit the mechanical activation of c-Kit induced by an anchored KitL. To do so, we carried-on dose-response studies with TKIs which
efficiently target c-Kit as well as other kinases which we suspect involved in c-Kit- and integrin-mediated spreading on fibronectin under KitL stimulation.
At first, we improve the screening test with TKIs. Hence, we proceeded to coat 96 wells to be able to test various concentrations of several molecules at the same time. We validated this format by examining again the effects of imatinib, as well as sunitinib and nilotinib. In using this set-up, we confirmed that imatinib doesn’t have any impact on immobilized-KitL mediated spreading. Under this condition, neither Nilotinib nor sunitinib blocked cell spreading at any of the tested doses. However, imatinib, sunitinib and to a lesser extent nilotinib efficiently reduced the percentage of MC9 spread cells.
The persistent adhesion observed after imatinib treatment is consistent with the adhesive response we find with COS cells transfected with the kinase-inactive mutant c-Kit D790N. The residue D790 functioned as a catalytic base in the transfer of a phosphate group from ATP and reduced the rate of degradation, internalization and receptor ubiquitination (Yee, Hsiau, et al., 1994). The c-Kit mutant D790N correspond to the dominant phenotype of the W42 allele (Ray et al., 1991) and results in an inactive kinase Nocka et al. 1990). In agreement with our results, the study of Dastych et al., demonstrated that W/Wv cells, (c-Kit kinase deficient) proliferate less but retain their capacity to adhere to fibronectin (Dastych et al., 1998). Also, W/Wv mastocytes could bind to cells expressing a membrane-bound form of KitL (Kinashi &
Springer, 1994). Accordingly, we showed that the m-KitL/c-Kit interaction is independent of the c-kit kinase activity (S. Tabone-Eglinger et al., 2014). Although these similarities, COS cells expressing c-KitD790N did not spread while imatinib-treated MC/9 cells did it. Thus, the necessity of a functional c-Kit kinase for an efficient cell spreading infers that the receptor must be phosphorylated. To note that as COS and MC/9 cells are of different origin, it is possible that these divergences result of a differential expression of integrins or their avidity for fibronectin induced by c-Kit.
As discussed in the introduction, imatinib, nilotinib, and sunitinib compete with ATP for binding to tyrosine kinase receptors. Like imatinib, Nilotinib targets the Abl kinase when inactive (Manley et al., 2005) but with higher affinity while it seems to preserve the same activity against c-Kit (Weisberg et al., 2006). Sunitinib prefers the inactive conformation but does not insert deep into the catalytic pocket (Roskoski, 2016). It has been shown that Nilotinib inhibits both c-Kit WT and the D814V variant (Gleixner et al., 2006; von Bubnoff et al., 2005). Indeed, it prevents the proliferation of BAF/3-c-KitD814V cells with an IC50 of
750nM (Ustun et al., 2011). Like our results, both imatinib and nilotinib do not have any effect in adhesion, migration, and engraftment of CD34+ cells at concentrations that block the cell surface expression of the VLA-4 and -5 but not CXCR-4 receptors (Belle et al., 2012). In the case of MC/9 cells, we do not know whether the treatment with such inhibitors will change the cell surface expression of integrins. It is possible that these drugs reduce the surface levels of integrins in the context of s-KitL stimulation. Further studies are required to correlate the c-kit kinase activity with the integrins expression or avidity and their implication in cell adhesion, spreading and migration.
Imatinib, nilotinib, and sunitinib have the same inhibition profile. All of them bind to the c-Kit receptor when the receptor is inactive (“DFG-out”). It means that the activation-loop block the substrate binding site thus inhibiting the catalytic activity of the enzyme. Imatinib, which was initially developed to target Abl mutations, efficiently block the wild-type c-Kit receptor as well as c-Kit mutants of the juxtamembrane region (Deininger et al., 2005; Mol et al., 2004).
However, it is less efficient in blocking c-Kit mutations within the A-loop which maintain the receptor in an active (“DFG-in”) state (DiNitto et al., 2010). This receptor state can be blocked by another group of inhibitors, notably dasatinib, providing a second line of treatments for imatinib-resistant cases (Tokarski et al., 2006). We, therefore, tested the effect of inhibitors targeting the active conformation (“DFG-in”) of kinases including dasatinib, bosutinib, ruxolitinib, and tofacitinib (Table 3). Among them, dasatinib was the only drug able to prevent cell spreading towards immobilized-KitL and fibronectin. Though in this condition, dasatinib inhibition still less (IC50~50nM) potent than the s-KitL-induced spreading (IC50~2nM).
Once again it seems that KitL presentation trigger different signaling pathways and cellular responses. Consequently, we proposed that in contrast to the soluble form, the immobilized-KitL might stabilize the c-Kit receptor in a fully-active conformation which cannot be targeted by imatinib. In such a structure, c-Kit allows the recruitment of intracellular adaptors which in turns might activate c-Kit kinase-independent signaling pathways leading to biological functions such as cell spreading. In this process, the SFKs, which are recruited to the phosphorylated-Y567 and activated might phosphorylate the Y900 residue of c-Kit (Lennartsson et al., 2003). Some of the SFKs members have been linked to c-Kit-mediated mast cells chemotaxis. For instance, activation of the FYN kinase leads to integrin-mediated Rac activation, cytoskeletal rearrangements and mast cells migration (Samayawardhena et
al., 2007). Besides, the SFKs can bind to the β subunit of integrins thus contributing to their activation and the control of cell spreading (Arias-Salgado et al., 2003; Reddy et al., 2008) Although with different affinities, dasatinib blocks a broad spectrum of protein kinase including c-Kit and kinases of the Src family. Does dasatinib inhibits cell spreading because of it can act on multiple targets? To rule out this hypothesis and point-out the involvement of Src as a possible point of convergence between c-Kit and integrins we used drugs with higher selectivity for the SFKs. Bosutinib and saracatinib neither affect s-KitL nor i-KitL-induced spreading and only showed a tendency to the inhibition at concentrations higher than 1µM.
Like dasatinib, bosutinib bind to the active conformation of Src. However, it appears to prefer the inactive conformation of Abl (Roskoski, 2016). Whether bosutinib binds to and inhibits c-Kit is sisolated kinase assays, as well as tumor cells invasion and paxillin phosphorylation in cancer cells (Green et al., 2009).
To summarize, dasatinib is the only TKI that inhibits the spreading of MC/9 cells in the context of an immobilized-KitL and sub-optimal fibronectin concentrations. In such a condition, the SFKs may connect c-Kit and integrins in mediating cell-matrix-spreading. Whether integrins are engaged before or after c-Kit is an open question. It is essential to understand this issue as the mechanism involved in the cooperation between cells and their microenvironment are likely responsible for the resistance to cancer treatments.