Validating novel RHOB partners in MAPK/ERK signaling in the context of anti-EGFR therapies – RHOBMAPK

Recent studies conducted in the team of Pr Gilles Favre at the CRCT have highlighted a potential role of the small GTPase RHOB in the resistance to Epidermal Growth Factor Receptor (EGFR) therapies. RHOB overexpression reduces cell sensitivity to erlotinib in a dose-dependent manner by restoring cell survival. In addition, our team has recently identified that activation of the RHO pathway drives the adaptive resistance to EGFR tyrosine kinase inhibitors (EGFR-TKi). The RHOBMAPK project aims to investigate the underlying mechanisms that control this response. Using our proprietary genetic split-GFP reporters, we have identified novel interacting partners of the small GTPase RHOB in the MAPK pathway, including the ERK kinase, which regulates essential cellular processes including cell proliferation and differentiation. We also showed that RHOB negatively regulates the activity of this pathway by decreasing ERK activity and cell viability. This newly discovered role of RHOB could be essential in the response to EGFR-TKi, as reactivation of ERK is a major cause of resistance to EGFR-TKi.
The project has been divided into three work packages and assigned to four research teams who specialize in the development of small GTPase signaling and biotechnological tools (CRCT-INSERM, Toulouse), proteomics (IPBS-CNRS, Toulouse), and biophysics/structural biology (IPBS-CNRS and TBI, Toulouse). Our first objective is to characterize the influence of RHOB post-translational modifications on the interactions with ERK in a PC9 bronchial adenocarcinoma model and to investigate how interactions can be modulated in response to tyrosine kinase inhibitors such as erlotinib and osimertinib. We will further examine the impact of RHOB expression on basal cellular homeostasis and during treatment with ERK and EGFR inhibitors. To gain a deeper understanding of the underlying mechanisms, we will examine the supramolecular arrangement of RHOB-ERK complexes using a combination of differential proteomic analysis of the associated complexes and the individual protein partners. In addition, we will conduct loss-of-function studies on proteins that are specifically linked to RHOB-ERK complexes in order to discover regulators of these complexes and identify phosphatases involved in the RHOB-dependent dephosphorylation of ERK. Our second objective concerns the biochemical, biophysical and structural analysis of RHOA-ERK and RHOB-ERK complexes. Preliminary proteomics analysis indicated that ERK1/2 isoforms can interact with RHOB and its homolog RHOA, thus confirming results obtained using our split-GFP technology. Considering the availability of previous NMR assignments of human RHOA and ERK2, we will compare the structure of both RHOA and RHOB in their GDP- and GTP-bound forms alone and in complex with ERK2. We will identify key residues potentially involved at the RHO-ERK2 interface and design mutants that can specifically reinforce these interactions for downstream structural studies. As a last objective, we will use biophysical information from RHOB-ERK2 mutants capable of destabilizing their interactions to explore their functional impact on cell homeostasis and signaling. We will evaluate the impact of knocking down regulatory proteins and expressing RHOB interaction deficient mutants on the cellular response to EGFR-TKi of EGFR mutated cell lines. The in vivo response to EGFR-TKI will be carried out with the most promising candidates to identify potential targets capable of increasing sensitivity to EGFR-TKi.