Project Summary Abstract Alteration of essential metabolic pathways is a major mechanism by which oncogenic KRAS promotes tumor development and growth in pancreatic ductal adenocarcinoma (PDAC). KRAS-driven PDAC is dependent on macropinocytosis (MP) and autophagy to fuel the high metabolic demand of rapid proliferation. Thus, these metabolic processes are attractive targets for the development of treatments for PDAC. Genetic depletion of KRAS results in downregulation of MP in PDAC. Additionally, our lab demonstrated that KRAS depletion or inhibition of ERK-MAPK signaling decreased glucose uptake and glycolysis but increased autophagy, thereby enhancing dependency on autophagy for essential nutrient supply. Accordingly, dual RAS-pathway and autophagy inhibition via chloroquine (CQ) synergistically enhanced efficacy in PDAC. Clinical data demonstrated that resistance to this treatment arises over time through unknown mechanisms. Preliminary data indicates that following RAS or ERK inhibition, both autophagy induction and MP downregulation are transient—with autophagic and MP activity returning to/surpassing basal levels after prolonged treatment. Recent work has suggested that these nutrient scavenging processes are able to compensate for loss of each other. We demonstrate an inverse temporal relationship between autophagy and MP following depletion or inhibition of the KRAS-ERK MAPK pathway. Furthermore, we show that CQ, commonly thought to inhibit lysosomal acidification, does not prevent degradation of MP-derived proteins. We hypothesize that there is compensatory regulation between autophagy and MP in the context of RAS-pathway inhibition. Thus, during prolonged RAS-pathway and autophagy inhibitor treatment, PDAC cells upregulate MP, consequently abrogating dependency on autophagy and reducing sensitivity to autophagy inhibition. Aim 1 will test the hypothesis that upregulation of MP confers resistance to dual autophagy and RAS-pathway inhibition in vitro and in vivo. Additionally, we present the unexpected observation that prolonged inhibition of the ERK-MAPK pathway results in the upregulation of MP. Previous PDAC studies have employed genetic depletion of KRAS to demonstrate the role of oncogenic KRAS in driving MP with no evaluation of inhibitors of KRAS or the ERK-MAPK pathway. Given the dependence of PDAC on macropinocytic uptake for tumorigenic growth, it is imperative to understand how these inhibitors regulate MP. Initial Aim 2 studies will be dedicated to comprehensive characterization of MP regulation in the context of genetic and pharmacological inhibition of KRAS and ERK over time. Subsequent work in Aim 2 will determine whether the changes in MP via RAS-pathway inhibition corresponds with changes in uptake and efficacy of nab-paclitaxel. Further understanding of RAS-driven regulation of these essential metabolic pathways in PDAC will inform future development of RAS-pathway inhibitor combinations. These studies will require my ap...