The concept of the niche was first used by Schofield in 1978 to describe a physiological microenvironment capable of regulating the fate of hematopoietic stem cells (Schofield, 1978). The niche integrates intrinsic and extrinsic factors that control the balance between self-renewal and differentiation as well as quiescence and proliferation of stem cells (Moore
& Lemischka, 2006). Among the most study niches is the hematopoietic niche. It includes cells (hematopoietic, stromal), soluble molecules (growth factors, chemokines), extracellular matrix proteins and physical components (shear stress, oxygen pressure) (Nardi & Alfonso, 1999). Within the hematopoietic niche, the fate of HSC is controlled by the expression of adhesion molecules, cytokines/chemokines and growth factors but also by extracellular matrix molecules. The interaction of the HSCs with other cells or extracellular matrix molecules are necessary for homing, maintenance and quiescence of HSC in the long term.
On the other hand, the niche would also serve as a stock of HSCs that can be released into the blood during mobilization or short-term hematopoietic reconstitution (Wilson & Trumpp, 2006).
Stromal cells are one of the major components of the hematopoietic niche. It is a heterogeneous population composed of endothelial cells, fibroblasts, adipose cells, macrophages and T lymphocytes. These cells secrete soluble factors (chemokines or cytokines) which are positive or negative external regulators of hematopoiesis (Zibara et al., 2012). For example, endothelial cells secrete the chemokine SDF-1, which through the interaction with the CXCR4 receptor on hematopoietic cells plays a crucial role in the in vivo and in vitro migration (homing) of CD34+ HSCs (Aiuti et al., 1997; Kollet et al., 2001). The Kit ligand (KitL) is an example of stimulating cytokine. It acts on its receptor c-Kit at the HSC level thus potentiating self-renewal, survival, and migration (Bowie et al., 2007; Kollet et al., 2006), as well as adhesion to the niche (Kovach et al., 1995). Therefore, the paracrine effect facilitated by the cytokines/chemokines represent the two primary mechanisms of regulation of the hematopoietic cells by their microenvironment. The ECM is another critical element of the niche. It constitutes a complex network of fibrous or non-fibrous molecules to which the HSCs adhere. This adherence is probably crucial, insofar as the matrix represents a considerable reservoir of growth factors. The major adhesive matrix proteins include
fibronectin (produced by endothelial cells and fibroblasts), osteopontin, collagen, thrombospondin, laminin, vitronectin or hyaluronic acid (Shiozawa et al., 2008; Taichman, 2005; Wilson & Trumpp, 2006). Among the adhesive proteins of the ECM, fibronectin offers the most reliable adhesion of HSCs to engineered coated surfaces (Franke et al., 2007). In vitro studies have shown that fibronectin can either induce or inhibit the proliferation of HSCs (Feng et al., 2006; Kramer et al., 1999). Hematopoietic progenitor cells can adhere to fibronectin via the integrins α4β1 and α5β1 (Muth et al., 2013). These integrins are also required for migration and homing of HSC within the niche (Papayannopoulou et al., 2001).
Integrins, as well as N-cadherins, mediate adhesion of HSCs to stromal cells (fibroblasts or osteoblasts). N-Cadherin is expressed by both HSCs and osteoblasts and appears to mediate HSC-osteolineage adhesion (Nakamura et al., 2010; J. Zhang et al., 2003). On the other hand, integrins like α4β1 and α5β1, expressed by HSCs, are essential for their anchorage to osteoblast and survival (Jung et al., 2005). Also, we showed in vitro that membrane-bound KitL could anchor mast cells expressing the c-Kit receptor (S. Tabone-Eglinger et al., 2014).
The cell microenvironment is perceived as a regulator in the development of malignant diseases (Le Bousse-Kerdiles, 2012). The notion of a hematopoietic niche can be transposed from normal to leukemic stem cells (LSCs) which can compete with regular HSCs for interactions within the niche. Thus, the niche microenvironment permits the maintenance and survival of LSCs (Zhou et al., 2016) and can also change the resistance of malignant cells to chemo and radiotherapies (Konopleva et al., 2002; Rich & Bao, 2007). LSCs express the α4β1 and α5β1 integrins which seem to be a poor prognostic. In fact, α4β1 allows the interaction of the blasts with the FN protecting the leukemic cells from apoptosis via the activation of the PI3K/Akt/Bad2 signaling pathway (Matsunaga et al., 2003). Besides, leukemic blasts have been shown to secrete and express more fibronectin at their surface (Vialle-Castellano et al., 2004), which has been reported to confer resistance to chemotherapy (CAM-DR for Cell Adhesion Mediated-Drug Resistance (Shain & Dalton, 2001). For more than ten years, leukemias has been treated with a therapeutic strategy that preferentially targets proliferating leukemic cells. However, this approach often spares the population of the LSCs (quiescent cells and cells expressing drug efflux proteins). Nowadays, an elemental concern is to develop drugs to target and kill LSCs while maintaining the regular quota.
It is now clear that the single transformation of HSC is not sufficient to induce leukemia, but the presence of a permissive tumor microenvironment to tumor growth and metastasis is also
necessary. Among the possible scenarios that can explain the contribution of the niche to the malignant transformation, we are interested in the adhesive interactions of hematopoietic cells, mainly the KitL/c-Kit and integrins axes. Since its discovery, the c-Kit structure, kinase activity as well as the physiological and its oncogenic role have been extensively studied. In the early 2000’s, targeted therapies raised with the development of a new class of small pharmacological molecules called tyrosine kinase inhibitors (TKIs). These drugs have revolutionized the treatment of cancers as they are more efficient in targeting and killing tumor cells while maintaining the normal quota than conventional chemotherapy. Some of these inhibitors targeted the c-Kit receptor and had significantly improved the management of GIST driven by a c-Kit activating mutation. Unfortunately, the molecules currently used in the clinic have some limits. They are not specific for a single kinase, and side effects are frequent in patients. Also, patients who initially respond to the treatment end up developing an acquired resistance to therapy. Other pathologies such as systemic mastocytosis, which is linked to the c-Kit D816V mutation, are treated only symptomatically. In fact, the inhibition of this c-Kit mutant still a challenge.
Therefore, we have focused this thesis on the study of the KitL/c-Kit oncogenic pathway using mutagenesis and pharmacological treatments. More specifically, we addressed the role of KitL and c-Kit in mediating cell-matrix adhesion and spreading on fibronectin. We also evaluated the differential responses to the soluble- or immobilized-KitL presentation as well as the role of the kinase activity. To achieve these goals, we have mainly used two cell lines:
MC/9 and COS cells. MC/9 cells are murine mast cells of hematopoietic origin which naturally express the tyrosine kinase receptor c-Kit. COS cells are fibroblast-like cells which lack c-Kit and KitL (Reith et al., 1991; Serve et al., 1994). Because MC/9 naturally express the murine c-Kit receptor we used them to study the function of c-c-Kit as well as other kinases in cell-matrix spreading. Because of the absence of c-Kit, we used COS cells as a model for murine c-Kit transient expression to functionally study different variants (WT or mutated) of c-Kit. A tagRFP, which locates the c-Kit, has been inserted in C-terminal. Different regions of c-Kit mutants were mutated and their effect on cell spreading assessed. To study the role of KitL and c-Kit in cell-matrix interactions we also made use of TKIs, mainly drugs that target c-Kit as well as proteins involved with this receptor.