Ubiquitin can be attached to proteins in the form of polymeric chains or as a single moiety; and the various ubiquitin modifications can lead to different functional outcomes. Our recent studies suggest that reversible ubiquitination of the RAS GTPases might alter their dimerization, attachment to the plasma membrane, and/or the interaction network, representing a novel mechanism of RAS regulation. To assess how ubiquitin conjugation affects RAS function, we will attach ubiquitin to specific lysines by combining synthetic biology and chemical ubiquitination approaches. We will determine the effect of ubiquitin conjugation on biochemical and interaction properties of RAS proteins. These studies will not only allow us to assess how RAS ubiquitination affects its interactions with known RAS interactors, but could also identify novel interactors that bind preferentially ubiquitinated RAS.
In our recent studies, we have uncovered the ubiquitin machinery controlling RAS ubiquitination. These studies indicate that disease-associated alterations in the ubiquitin machinery represent an alternative mechanism that drive RAS hyperactivation in human disease. The variety of pathological manifestations and frequency of genetic involvements represent rightful indicators for the breadth of phenotypic effects that could be associated with dysregulated RAS ubiquitination. We will continue to assess the functional impact of the RAS ubiquitination machinery in human disease using genetically modified mouse models. Specifically, we are generating Lztr1- and Traf7- knockout cancer models. In parallel, we are performing structural characterization of the disease-associated mutants of LZTR1 and TRAF7. The generated disease models will provide us a useful platform to delineate pathways involved in RAS-driven disorders and could be used as preclinical models for drug validation in subgroups of patients with specific genomic alterations.
To unravel the full extent of the ubiquitin network involved in control of RAS ubiquitination, we will screen for the components of the RAS ubiquitination machinery by performing the NanoBRET biosensor-based targeted CRISPR screen. Using mass-spectrometry and conventional biochemical approaches, we will assess whether the identified enzymes are involved in ubiquitination of the RAS GTPses. The identified enzymes could represent potential targets to inhibit RAS activity.
OTUB1, a negative regulator of RAS ubiquitination, is commonly amplified and overexpressed in lung cancers.We demonstrated that suppression of OTUB1 inhibits the xenograft growth of lung tumors, suggesting that OTUB1 could be a promising target for anti-cancer therapy. Given that OTUB1 inhibits RAS ubiquitination independently of its catalytic deubiquitinase activity by sequestrating of E2 ubiquitin-conjugating enzymes charged with ubiquitin. We are also screening for inhibitors that block activity the deubiquitinase OTUB1, a negative regulator of RAS ubiquitination. Thus, our aim is to identify compounds that block the interaction between ubiquitin or ubiquitin-charged E2 and OTUB1. In the initial screen, we have identified two compounds that block OTUB1 activity in a micromolar range. Currently, we plan to perform several rounds of optimization to improve pharmacological properties of OTUB1 inhibitors. Using in vitro and in vivo models, we will then perform anti-tumour properties of the identified compounds. The identified compounds could represent a novel therapeutic strategy for RAS-associated diseases.
