Regulation of Nutrient Stress-Induced Macropinocytosis in Pancreatic Ductal Adenocarcinoma

PROJECT Summary: The world has been shocked by the recent COVID-19 pandemic. Macropinocytosis is a form of endocytosis thatviruses use to gain entry into cells or to facilitate infection. Our laboratory has the most extensive experience ininvestigating macropinocytosis in the context of cancer, where it functions as a nutrient acquisition pathway2-12.Macropinocytosis is unique among other endocytic pathways because it is preceded by plasma membraneactivity in the form of ruffling. When ruffles fuse with each other they form a macropinosome that encapsulatesthe surrounding fluid and associated particles. Viruses can use macropinocytosis for cellular internalization orthey can hijack macropinocytosis for other aspects of infection. Severe acute respiratory syndromecoronaviruses (SARS-CoVs) induce macropinocytosis late in infection that is continuous, independent from cellentry, and associated with increased infection in vitro. These viruses use their Spike protein to signal throughthe epidermal growth factor receptor (EGFR) to stimulate macropinocytosis, and inhibitors of macropinocytosisor EGFR lead to a reduction in viral spread. While CoVs do not seem to utilize macropinocytosis for entry intothe cell, they do use later steps in the macropinosome maturation pathway to augment infection. The maturationof the macropinosome is not very well characterized, but there are some indications that specific signalingpathways are involved. We have extensively studied EGFR-driven macropinocytosis in cancer cells and we canuse this expertise to determine strategies for blocking macropinosome maturation and SARS-CoV-2 infection.Our hypothesis is that SARS-CoV-2 uses mature macropinosomes as a way to spread to surrounding cells,either by inducing `macropinosome bursting' as occurs in a process called methuosis, or by travelingextracellularly via recycling macropinosomes. In this proposal, we will explore this hypothesis by 1) taking acandidate approache to blocking macropinosome maturation and 2) determine the mechanism of SARS-CoV-2viral escape that involves macropinocytosis. This project will be of great significance and impact because, byand large, a detailed picture of what controls macropinosome maturation in the context of EGFR signaling andSARS-CoV-2 is lacking. Moreover, it will constitute the first evaluation of the impact that candidate inhibitorshave on macropinosome maturation. Understanding the regulators of macropinosome maturation will shed lighton a critical aspect of CoV biology and could lead to new approaches for the treatment of COVID-19.