Systems analysis of the spatial regulation of oncogenic Ras signalling

Abstract

Ras (Rat sarcoma) isoforms are small GTP-binding proteins that play a major role in the signalling networks controlling cell growth and survival. The Kras isoform is of particular interest as in many severe kinds of cancer the presence of oncogenic Kras mutations is associated with a poor prognosis. Kras is associated with the plasma membrane due to its farnesyl moiety and a polybasic motif and functions as a signalling hub. If Kras gets lost from the plasma membrane due to spontaneous dissociation or endocytosis, it will equilibrate over the extensive endomembrane system inside the cell. With Kras no longer present at the plasma membrane, its activation and the following activation of subsequent pathways can no longer take place. However, to remain on the plasma membrane, Kras has to be constantly enriched there. This enrichment must be actively maintained in the cell by an energy-driven mechanism involving the solubilising factor PDEδ. Consequently, inhibition or down-modulation of PDEδ results in mislocalisation of Kras, making PDEδ an interesting target for anti-cancer drug development. In 2013 a small molecule, Deltarasin, was identified as a potent inhibitor of PDEδ causing a redistribution of Kras from the plasma membrane towards endomembranes when applied to cells. This work investigates whether small molecule PDEδ inhibitors such as Deltarasin affect (K) Ras localization in space and time. It demonstrates that PDEδ inhibition causes Ras relocalisation from the plasma membrane towards the endomembranes in different human cancer cell lines and in murine small intestine organoids, which express endogenous levels of oncogenic Kras. Nonetheless, it has been shown that Deltarasin has certain side effects, e.g. it becomes cytotoxic at higher concentrations. Hence, a new PDEδ inhibitor, Deltazinone 1, which is supposed to be less cytotoxic in comparison to Deltarasin, was synthesized. In order to determine whether it represents a viable alternative to Deltarasin, its ability to relocalise Kras in a panel of cancer cell lines was tested and it was indeed possible to mislocalise Kras with this inhibitor in Kras-dependent PancTuI cells. Deltazinone 1 and Deltarasin both had a demonstrable effect on cell growth/survival, respectively. In this way it appears that PDEδ constitutes a valid target for the pharmacological therapy of Kras-dependent tumours. This work demonstrates that two specific PDEδ inhibitors with completely different lead structures are capable of mislocalising Kras to endomembranes. In sum, it demonstrates that the availability of PDEδ is essential to ensure (K) Ras localization at the plasma membrane in Kras-dependent cancer cells and thus that the survival of those cells are ultimately dependent on PDEδ.