Objective 1. Reconstruct the network topology and connection strengths in the Survival-Apoptotic-Mitogenic (SAM) network. The network reconstruction method called Modular Response Analysis (MRA) was improved in two ways. First, it was reformulated to take into account effects of protein sequestration, which are inevitable in enzyme regulatory networks. Second, the robustness of MRA was tested with respect noise in input data and the optimal strategy of analysis of noisy data was suggested. Third, based on proteomics data obtained from collaborators, we assessed how mutant oncogenic RAS rewires intracellular signalling. The developed model of network rewiring was in agreement with available experimental data.
Objective 2. Develop site-specific dynamical models of SAM network. To achieve objective 2 we have developed a number of structure-based ODE models using rule-based modelling technique that predict whole-network responses to different types of kinase inhibitors. Kinase inhibitors were classified according to the positions of the DFG motif (DFG-IN/OUT) and a structurally conserved alpha C helix (αC-IN/OUT). The model of MAPK pathway showed that combinations of two RAF inhibitors of different types can synergistically inhibit signalling by RAF family kinases, while each inhibitor on its own is ineffective, especially in RAS-mutant tumours. The computational model of ErbB-related signalling predicted that combinations of two ErbB inhibitors of different types can synergistically inhibit phosphorylation and downstream signalling of ErbB family tyrosine kinase receptors. The computational model of JAK-related signalling suggested that combinations of two types of JAK inhibitors will show synergy in inhibition of the JAK-STAT signalling pathway in cells resistant to JAK inhibitor therapy. In all 3 cases exact types of inhibitors producing synergism were predicted by a mathematical model and depended on mutational and expression background.
Objective 3. Test the predictions of mathematical modelling of SAM network. To achieve objective 3 we performed a number of signalling, proliferation and colony formation experiments to prove the existence of drug synergy effects predicted by computational models. The experiments in RAS and RAF mutant melanoma cell lines, HER2-positive breast cancer cell lines and T-ALL cell line corroborated model predictions on drug synergy between different types of RAF, ErbB and JAK inhibitors. Thus, all key model predictions were validated experimentally.
Based on achieved results, 4 original research papers and 2 reviews were published, and 1 patent application was filed.
