Periodic Reporting for period 3 - UB-RASDisease (The ubiquitin system in RAS-driven disease) Reporting period: 2021-04-01 to 2022-09-30 Summary of the context and overall objectives of the project The RAS pathway is the most frequently activated signalling node in human disease. Despite intensive efforts, effective therapeutic strategies for RAS-driven disease remain daunting. Our recent studies show that reversible ubiquitination represents a conceptually novel mechanism of RAS regulation. During our recent studies, we have partially uncovered the molecular machinery controlling ubiquitination of the RAS protein network. Strikingly, loss of function of positive regulators of RAS ubiquitination, LZTR1 and TRAF7, is associated with a broad range of human disease, whereas a negative regulator of RAS ubiquitination, OTUB1, is commonly amplified and overexpressed in wild-type RAS epithelial cancers. Thus, the genetic evidence and our initial functional data explicitly point out to the contribution of the ubiquitin system to the pathogenesis of RAS driven diseases. Understanding mechanisms of RAS proteostasis will not only allow us to identify patients who might benefit from the RAS pathway inhibitors, but will likely lead toward novel therapeutic approaches for these patients. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Despite of constant and exhaustive efforts to characterize RAS proteins, LZTR1 is the first novel RAS regulator, implicated in human diseases, that has been identified since years. Multiple genetic studies overwhelmingly point out for the role of LZTR1 in a wide range of human disorders, such as Noonan Syndrome (a genetic disease), liver cancer, childhood cancer, and Schwannoma, a benign tumor that affects nerves. Our functional studies confirmed that LZTR1 contributes to human diseases by acting as a part of the ubiquitin ligase complex that mediates conjugation of ubiquitin to RAS proteins. This conjugation reduces RAS recruitment to the membrane and thus its activation and downstream signalling. We hope that the discovery of this alternative mechanism of RAS regulation will lead toward novel therapeutic approaches for RAS-driven diseases. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) 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.
