TSC-insensitive Rheb mutations induce oncogenic transformation through a combination of constitutively active mTORC1 signalling and proteome remodelling.

The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cellular metabolism that is commonly hyperactivated in cancer. Recent cancer genome screens have identified multiple mutations in Ras-homolog enriched in brain (Rheb), the primary activator of mTORC1 that might act as driver oncogenes by causing hyperactivation of mTORC1. Here, we show that a number of recurrently occurring Rheb mutants drive hyperactive mTORC1 signalling through differing levels of insensitivity to the primary inactivator of Rheb, tuberous sclerosis complex. We show that two activated mutants, Rheb-T23M and E40K, strongly drive increased cell growth, proliferation and anchorage-independent growth resulting in enhanced tumour growth in vivo. Proteomic analysis of cells expressing the mutations revealed, surprisingly, that these two mutants promote distinct oncogenic pathways with Rheb-T23M driving an increased rate of anaerobic glycolysis, while Rheb-E40K regulates the translation factor eEF2 and autophagy, likely through differential interactions with 5' AMP-activated protein kinase (AMPK) which modulate its activity. Our findings suggest that unique, personalized, combination therapies may be utilised to treat cancers according to which Rheb mutant they harbour.

Constitutively active Rheb mutants [T23M] and [E40K] drive increased production and secretion of recombinant protein in Chinese hamster ovary cells.

Monoclonal antibodies (mAbs) are high value agents used for disease therapy ("biologic drugs") or as diagnostic tools which are widely used in the healthcare sector. They are generally manufactured in mammalian cells, in particular Chinese hamster ovary (CHO) cells cultured in defined media, and are harvested from the medium. Rheb is a small GTPase which, when bound to GTP, activates mechanistic target of rapamycin complex 1, a protein kinase that drives anabolic processes including protein synthesis and ribosome biogenesis. Here, we show that certain constitutively active mutants of Rheb drive faster protein synthesis in CHO cells and increase the expression of proteins involved in the processing of secreted proteins in the endoplasmic reticulum, which expands in response to expression of Rheb mutants. Active Rheb mutants, in particular Rheb[T23M], drive increased cell number under serum-free conditions similar to those used in the biotechnology industry. Rheb[T23M] also enhances the expression of the reporter protein luciferase and, especially strongly, the secreted Gaussia luciferase. Moreover, Rheb[T23M] markedly (2-3 fold) enhances the amount of this luciferase and of a model immunoglobulin secreted into the medium. Our data clearly demonstrate that expressing Rheb[T23M] in CHO cells provides a simple approach to promoting their growth in defined medium and the production of secreted proteins of high commercial value.