Regulation of KRAS plasma membrane targeting by defined glycosphingolipids.

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PROJECT SUMMARY KRAS operates as a molecular switch that oscillates between an active GTP-bound and inactive GDP-bound state. Active KRAS proteins are required to localize to the plasma membrane within a defined lipid environment to be functional. Oncogenic KRAS induces metabolic dependence on aerobic glycolysis, a phenomenon known as the Warburg effect. We recently discovered that aerobic glycolysis, by generating a subset of outer leaflet glycosphingolipids (GSL), controls KRAS plasma membrane (PM) interactions and therefore KRAS oncogenesis. Blocking GSL metabolism, using multiple methods including glucose deletion, pharmacological inhibition and genetic deletion of selected GSL synthetic enzymes all mislocalize KRAS from the PM. Deletion of key enzymes of the GSL biosynthetic pathways abrogated KRAS oncogenesis in vitro and in vivo. Building on these exciting findings, we developed our central hypothesis: that a subset of defined glycosphingolipids regulates localization and nanoscale organization of KRAS through controlling the lipid composition on the PM and lipid transport at PM-ER membrane contact sites. We will test to what extent do GSLs operate as spatial organizers of the inner PM and ER-PM contacts sites in Aim1 by using a variety of lipid biosensor and high- resolution imaging. In Aim 2, we will employ genetic approaches, lipid reconstitution experiments and molecular dynamics simulation to probe the potential mechanism of inter leaflet coupling between outer leaflet GSLs and inner leaflet phosphatidylserine on the PM. Finally, in Aim 3 we will rigorously test the relevance of this novel glycosphingolipid-centered mechanism in multiple mouse KRAS cancer models. In sum, using a multidisciplinary approach, this proposal will yield new insights into the molecular mechanism of how surface GSLs regulate PM lipid composition and lipid transport, and the role of inter leaflet lipid coupling. This is highly significant to basic membrane biology and biophysics. The connection of GSL metabolism through PM lipid organization to KRAS function will identify a novel vulnerability to KRAS oncogenesis that may have therapeutic potential.