Functional Perturbation of the Gab2 Multiprotein Complex by High-affinity SH2-binding Monobodies in Chronic Myelogenous Leukemia
Chronic myeloid leukemia is characterized by a reciprocal chromosomal translocation between chromosome 9 and 22, resulting in the expression of the Bcr-Abl oncoprotein. Despite the great improvement in patient survival using tyrosine kinase inhibitors (TKI), resistance caused by point mutations in the Abl kinase domain is a major drawback in therapy. Bcr-Abl forms a multi-protein complex and Gab2 is one of the few functionally critical scaffold proteins and substrates of Bcr-Abl. The goal of this thesis was to understand the contributions of the different pathways activated downstream of Gab2. In order to pursue this aim, we aimed at targeting the Gab2 protein complex with high-affinity and highly specific engineered fibronectin type III monobodies, which act as small protein interaction inhibitors, binding to the SH2 domains of Gab2 interactors. Firstly, the Gab2 complex was studied in an unbiased way by using interaction proteomics in order to confirm the pre-defined interactors of Gab2 and find out new interactors that could be possibly targetable by monobodies. To this end, Gab2 and the known interactors were affinity-tagged and expressed in cells and interacting proteins were identified after tandem affinity purification by protein mass spectrometry. Many known interactors were confirmed and possible novel interactors were identified. Next, the SH2 domains of the Gab2 interactors SHP2 and PLCÎ³1 were targeted by monobodies and binding clones were functionally characterized. Initially, monobodies targeting the SH2 domains of SHP2, which is an oncogenic tyrosine phosphatase, were studied. Tandem affinity purification of the monobodies demonstrated that they are monospecific for their cognate target protein and no other SH2 domain-containing proteins were identified. Upon monobody expression in cell lines, we were able to show a significant decrease of phosphorylation of tyrosine residues of SHP2 that are critical for its catalytic activity. Monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Gab2 scaffold protein, suggesting the decoupling of SHP2 from the Bcr-Abl protein complex by disrupting the phosphotyrosine-SH2 mediated interaction. Moreover, SHP2 downstream signaling was altered upon monobody expression. HCC-1171 non-small cell lung cancer cell line was investigated for ERK phosphorylation. Strikingly, ERK phosphorylation was abolished in the HCC-1171 cells upon monobody expression. The physiological effects of monobodies were investigated in an inducible expression system in HCC-1171 cells. However, we could not detect significant changes in cell proliferation or ERK phosphorylation when compared to the non-binding control monobody. Furthermore, tandem monobodies targeting both SH2 domains of SHP2 simultaneously resulted in decreased ERK phosphorylation in Bcr-Abl overexpressing cells. PLCÎ³1 targeting monobodies were also monospecific for their target protein in cells. In addition, PLCÎ³1 phosphorylation was decreased upon monobody expression in cells. Although monobodies against p85 subunit of PI3K remains to be evaluated and better monobodies are required for PLCÎ³1 targeting, our results validate monobodies as potent and specific antagonists of protein-protein interactions in cells and they could be powerful tools to investigate the participation of different signaling pathways in malignant transformation.
Programme doctoral Biotechnologie et Génie biologique
Faculté des sciences de la vie
Institut suisse de Recherche Expérimentale sur le Cancer
Chaire Fondation ISREC en oncologie translationnelle
Public defense: 2015-3-30
Record created on 2015-03-23, modified on 2016-08-09