Abstract A14: Mutations in the ubiquitin ligase adaptor LZTR1 drive human disease by dysregulating RAS ubiquitination and signaling

Abstract

Abstract Genetic evidence explicitly points out a role for LZTR1, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, in human disease. Mutations in LZTR1 concurrent with loss of heterozygosity have been associated with glioblastoma and schwannomatosis. LZTR1 emerges as a predisposition gene for paediatric neoplasms. LZTR1 mutations are significantly higher than the background in liver cancers and testicular germ cell tumours. LZTR1 has been also recently added to the list of genes causing Noonan Syndrome, a RASopathy disorder. However, how LZTR1 contributes to human disease and whether it regulates the RAS/MAPK pathway are not known. We found that whereas Lztr1 knockout in mice is embryonically lethal, Lztr1 haploinsufficiency partially recapitulates Noonan syndrome phenotypes, including growth delay, craniofacial dysmorphism, and congenital heart defects. On the other hand, LZTR1 loss in primary Schwann cells drives their dedifferentiation into proliferating cells. By trapping LZTR1 complexes from intact mammalian cells, we identified RAS as a substrate for the LZTR1/CUL3 ubiquitin ligase complex. Immunoprecipitation experiments confirmed that endogenous LZTR1 is associated with endogenous RAS. Ubiquitome and proximity ligation analyses showed that Lztr1 loss abrogates ubiquitination of endogenous RAS specifically at lysine 170. Molecular simulations together with the analysis of Hras-K170R knock-in cells demonstrated that LZTR1-mediated ubiquitination at lysine 170 inhibits RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations abolish RAS ubiquitination due to disruption of either the LZTR1/CUL3 complex formation, or the interaction with RAS proteins. In line with the work of Bigenzahn et al., Giulio Superti-Furga lab, CeMM (see separate abstract), our study implies LZTR1-mediated RAS ubiquitination as a key step in the control of the RAS GTPases. The discovered molecular mechanism provides a fundamental explanation for the role of LZTR1 in human disease. Citation Format: Mikhail Steklov, Silvia Pandolfi, Maria Francesca Baietti, Paolo Carai, Mingzhen Zhang, Hyunbum Jang, Yanyan Cai, Eric Legius, Jan Tavernier, Kris Gevaert, Francis Impens, Stephane Heymans, Ludwine Messiaen, Ruth Nussimov, Sven Eyckerman, Anna Sablina. Mutations in the ubiquitin ligase adaptor LZTR1 drive human disease by dysregulating RAS ubiquitination and signaling [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A14.