Regulation and tumorigenesis of non-classical RAS oncoproteins - Resubmission - PROJECT SUMMARY Oncoproteins drive tumor development and aggressive growth by dysregulating the signaling pathways involved in cell growth, survival, and proliferation. Targeting oncoproteins directly, or their downstream pathways, has resulted in novel therapies currently being used for the treatment of different cancer subtypes. The RAS family of guanosine triphosphatase (GTP) hydrolases (GTPases) are a group of oncoproteins, that have roles in cancer initiation, progression, and resistance to therapies. Furthermore, mutations in genes encoding classical RAS proteins are very frequent in cancer. RAS proteins cycle between a guanosine diphosphate (GDP) inactive form and a GTP active form. In its GTP-bound conformation, RAS proteins promote activation of downstream signaling pathways that contribute to the growth and proliferation of cells. This nucleotide cycle is assisted by accessory proteins that catalyze the loading or hydrolysis of GTP and dysregulation of this mechanism is also frequent in cancer. Emerging evidence has shown that non-classical RAS proteins also play critical roles in cancer biology; however, their mechanisms of regulation and tumorigenesis are not well-understood and differ from classical RAS proteins. We have recently discovered a novel mechanism of regulation of the non-classical RAS proteins RIT1 and MRAS that is mediated by ubiquitination and proteasomal degradation of the inactive GDP-bound form. Our work identified LZTR1, a novel protein that binds RIT1 and MRAS and forms a complex with the E3 Ubiquitin ligase Cullin-3 (CRL3LZTR1). Cancer-associated mutations in RIT1 or LZTR1 disrupt their interaction and lead to the accumulation of non-classical RAS GTPases and activation of downstream signaling. In this application, we propose to study critical aspects regarding the regulation of this novel complex, its contribution to tumorigenesis, and potential pharmacological strategies to restore protein degradation. In Aim 1, we will use biochemical and structural approaches to understand the determinants of LZTR1 selectivity towards the non-classical RAS GTPases RIT1 and MRAS and the function of its protein domains. In Aim 2, we plan to elucidate the signaling mechanisms that regulate CRL3LZTR1 activity in cells and how these pathways can contribute to the resistance to specific targeted therapies. In Aim 3, we will develop novel mouse models that are driven by an oncogenic RIT1 mutation that is resistant to LZTR1-mediated proteasomal degradation and analyze the molecular signatures of these tumors. In addition, we will develop proof-of-concept pharmacological treatments for RIT1-driven cancers. Overall, our project will address a major knowledge gap in the mechanisms of regulation of non-classical RAS oncoproteins and will provide novel preclinical models and therapeutic strategies for these cancers. The work described in this application is the first step to develop mechanism-based and precise medicines for targeting these oncogenic pathways in cancer.
