Mechanisms of Nonstop Extension Mutations in Tumor Suppressor Genes

• Applicant: Professor Dr. Sven Diederichs
• Subject Area: General Genetics and Functional Genome Biology
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 397982491

Cancer is a genetic disease for which missense and nonsense point mutations or copy number alterations have been studied as causative genetic changes.For the preceding project, we report the discovery of the functional relevance of the class nonstop mutations, which had never been studied in cancer. These mutations convert a stop codon into a regular codon adding a C-terminal extension to the protein. We have generated an extensive database of 3412 nonstop mutations in cancer (NonStopDB). Among cancer-related genes, the tumor suppressor SMAD4 was most frequently affected by nonstop mutations. Strikingly, nonstop mutations diminished SMAD4 protein expression in exogenous and endogenous precision genome editing models. We identified a transferable ten amino acid degron motif containing hydrophobic bulky residues that induces SMAD4 ubiquitination and proteasomal degradation (Dhamija, Nat Cell Biol 2020).Moreover, our preliminary data identified recurrent nonstop mutations in the tumor suppressors VHL and BAP1 linked to kidney cancer. These also diminished protein expression - but with notable differences to SMAD4: the VHL mutation only affected expression in the endogenous setting, but not upon overexpression, while its extension was also enriched for bulky residues. The extension of BAP1 was very long (205 aa) and did not contain a stretch of bulky residues.Thus, we hypothesize that nonstop mutations have a so far unexplored role in cancer genes and that C-terminal extensions and sequence motifs can generally control protein abundance.In the proposed project, we pursue three aims in three work packages (WP): In WP1, we explore the functional relevance and the molecular mechanism of the nonstop mutations in VHL with a special emphasis on the discrepancy between exogenously and endogenously introduced mutations. In WP2, we elucidate the mechanistic impact of nonstop mutations on BAP1 and also map sequence motifs in the long extension. In the largest WP3, we systematically analyze all nonstop mutations found in cancer for their impact on reporter protein expression. This will not only identify individual nonstop extensions with a strong impact on expression, but also enable analyses of C-terminal sequence motifs correlating with protein abundance, which will be further studied. Notably, we already found significant differences in the amino acid composition of the nonstop extensions versus the proteome in silico with an enrichment of bulky amino acids and a depletion of acidic residues.In summary, the proposed project will elucidate in detail the molecular mechanisms of individual nonstop mutations in the important tumor suppressor genes VHL and BAP1, and also systematically analyze the impact and motifs in all cancer-specific C-terminal extensions controlling protein expression levels. Importantly, the WPs can be pursued synergistically and benefit from shared methodologies and model systems, but all aims can also be achieved independently.

• DFG Programme: Research Grants