Ras GTPases represent a cancer-driving nexus that is difficult to target, underscoring the need to better characterize the spectrum of Ras-interacting biomolecules that govern its function. Using new methods designed to find previously missed interactors, recent CA142635 efforts defined the Ras RNA and protein interactomes to identify a functionally important new RNA binding capacity for Ras as well as 38 new cancer-relevant Ras-interacting proteins. Ras selectively and directly bound C/D box small non-coding RNAs snoRNAs, including SNORD50A/B, which potently inhibited Ras and was deleted at high frequency co-occurantly with RAS gene mutation in human cancer. Live-cell vicinal protein labeling followed by mass spectrometry detected protein interactions missed in prior studies, among which were mTOR and the putative palmitoyltransferase, ZDHHC5. This competing renewal will define the function of these new Ras interacting RNA and proteins in Ras-driven tumorigenesis. First, we will further characterize SNORD50A/B snoRNA impacts on Ras as well as the role of other newly discovered Ras-binding snoRNAs. Based on the finding that SNORD50A/B snoRNAs block farnesyltransferase binding to Ras, we will define the global impact of SNORD50A/B deletion on Ras protein-protein interactions to shed further light on the newly discovered modulation of Ras by RNA. We will also systematically test the function of additional Ras-binding RNAs on Ras-driven neoplasia, beginning with 6 snoRNAs that are recurrently mutated at high frequency in human cancer.
Aim I will test the hypotheses that SNORD50A/B snoRNAs remodel the Ras protein interactome to regulate Ras signaling and that additional Ras-binding snoRNAs alter Ras action in tumorigenesis. Second, we will characterize the newly identified Ras-mTOR interaction in tumorigenesis and will define the functional importance of additional vicinal proteins to oncogenic Ras function. We will define the domains on Ras isoforms and mTOR responsible for the observed direct binding of Ras to mTOR, examine the impact of Ras-mTOR binding on mTORC1 vs mTORC2 kinase activity and assess the impacts of disrupting Ras-mTOR binding on Ras- driven tumorigenesis in vivo. We will also systematically delete 38 newly identified Ras proximal proteins and quantify their impact on Ras-driven tumorigenesis.
Aim II will test the hypotheses that Ras-mTOR binding is functional in Ras-driven tumorigenesis and that multiple newly identified Ras-interacting proteins impact Ras signaling outcomes in neoplasia. At the end of the proposed funding cycle, this effort will define the function of newly identified Ras-binding RNAs and Ras-proximal proteins in Ras-driven tumorigenesis. 1

Public Health Relevance

REGULATORS OF TUMORIGENESIS Project Narrative Dominant oncogenes, such as Ras, drive tumorigenesis through an incompletely known web of interactions. We recently identified new RNAs and proteins that interact with Ras in cancer. Characterizing these Ras interactors is designed to provide new targets for cancer therapy. 1

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA142635-06
Application #
9175363
Study Section
Cancer Molecular Pathobiology Study Section (CAMP)
Program Officer
Watson, Joanna M
Project Start
2010-12-10
Project End
2021-07-31
Budget Start
2016-08-01
Budget End
2017-07-31
Support Year
6
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Stanford University
Department
Dermatology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
Siprashvili, Zurab; Webster, Dan E; Johnston, Danielle et al. (2016) The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer. Nat Genet 48:53-8
Zarnegar, Brian J; Flynn, Ryan A; Shen, Ying et al. (2016) irCLIP platform for efficient characterization of protein-RNA interactions. Nat Methods 13:489-92
Ungewickell, Alexander; Bhaduri, Aparna; Rios, Eon et al. (2015) Genomic analysis of mycosis fungoides and S├ęzary syndrome identifies recurrent alterations in TNFR2. Nat Genet 47:1056-60
Webster, Dan E; Barajas, Brook; Bussat, Rose T et al. (2014) Enhancer-targeted genome editing selectively blocks innate resistance to oncokinase inhibition. Genome Res 24:751-60
Lee, Carolyn S; Bhaduri, Aparna; Mah, Angela et al. (2014) Recurrent point mutations in the kinetochore gene KNSTRN in cutaneous squamous cell carcinoma. Nat Genet 46:1060-2
Jameson, Katherine L; Mazur, Pawel K; Zehnder, Ashley M et al. (2013) IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase-driven tumors. Nat Med 19:626-630
Kretz, Markus; Siprashvili, Zurab; Chu, Ci et al. (2013) Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 493:231-5
Kretz, Markus; Webster, Dan E; Flockhart, Ross J et al. (2012) Suppression of progenitor differentiation requires the long noncoding RNA ANCR. Genes Dev 26:338-43
Flockhart, Ross J; Webster, Dan E; Qu, Kun et al. (2012) BRAFV600E remodels the melanocyte transcriptome and induces BANCR to regulate melanoma cell migration. Genome Res 22:1006-14
Siprashvili, Zurab; Webster, Dan E; Kretz, Markus et al. (2012) Identification of proteins binding coding and non-coding human RNAs using protein microarrays. BMC Genomics 13:633

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