Identifying novel regulators of KRAS translation - PROJECT ABSTRACT: KRAS is a central oncogenic driver of pancreatic ductal adenocarcinoma (PDAC), the third leading cause of cancer mortality in the United States. KRAS dosage is critical to driving tumorigenesis; however, how oncogenes are translationally regulated to drive dosage remains poorly understood. The RNA levels of key oncogenic drivers often do not correlate with protein abundance, emphasizing the importance of translational control. The 5´ untranslated regions (UTR) of oncogenic mRNAs possess cis-elements, such as specific sequences and structures that, in normal cells, keep translation tightly regulated to maintain accurate protein dosage. However, this process is broken in cancer. KRAS is a key example of this, as it possesses a highly structured 5´UTR, yet which specific RNA binding proteins (RBP) interact with its 5´UTR and how they influence its translation efficiency, as well as how cancer cells exploit this process to promote KRAS dosage, are all areas that are still largely unexplored. To address this outstanding question, we used a selective translational reporter coupled with genome-wide CRISPRi screening and fluorescence-activated cell sorting to identify regulators of KRAS translation through its 5´UTR in human PDAC cells. For this proposal, we are exploring the top two activators identified in the screen: Upstream of N-Ras (UNR) and its interacting protein (UNRIP). UNR is an RBP that bridges UNRIP with target mRNAs; however, little is known about the mechanism of action and neither have been previously reported to regulate the KRAS mRNA or PDAC. I hypothesize that UNR and UNRIP promote KRAS translation through the 5´UTR to favor PDAC cell growth and survival. We found that the depletion of UNR and UNRIP decreases the translation and protein levels of KRAS. In Aim 1, we propose to determine the mechanism by which UNR and UNRIP promote KRAS translation through the 5´UTR using polysome profiling, luciferase assays, translation initiation complex formation assays, and cross-linked immunoprecipitation sequencing. In Aim 2, we will define the role of UNR and UNRIP in driving PDAC through KRAS translation in vitro and in vivo. We found a decrease in the colony formation capacity of PDAC cells upon UNR and UNRIP depletion, which was dependent upon KRAS expression. To further explore these compelling results, we will use a range of cell viability assays across various PDAC models that range in KRAS dependency. Additionally, we will test the therapeutic relevance of this regulation using RAS signaling inhibitors and in vivo mouse models. Altogether, this project will determine how PDAC coordinates the selective translation of KRAS through a novel mechanism of post-transcriptional gene regulation, ultimately leading to the discovery of selective avenues for therapeutic intervention in cancer. The proposed research will be conducted in the scientifically rich environment of UCSF under the mentorship of an expert in the field. There is a rigorous training plan in place, including a wide breadth of scientific concepts and techniques across RNA biology and cancer, and cultivation of science communication and mentorship skills, ultimately to pursue a career as an independent investigator.
