DESCRIPTION (provided by applicant): The central goal of this proposal is to understand how sphingosine kinase 1 (SK1) is regulated in order to build better inhibitors as alternative cancer therapeutics. SK1 is an important enzyme in the sphingolipid metabolism pathway as it sits between the pro-apoptotic sphingolipids ceramide and sphingosine and the pro- survival and pro-angiogenic lipid sphingosine-1-phosphate (S1P). S1P has been shown to have both intra- and inter-cellular signaling properties that play an important role in angiogenesis and invasion. SK1 has been detected in numerous different cancer types and cell lines. Additionally, SK1 activity has been shown to be over-expressed in several different cancer types. SK1 activity has been previously shown to be modulated by anionic phospholipids (APLs) which can be found in the plasma membrane of all cells. Furthermore, it has been shown that SK1 can translocate to the plasma membrane where its substrate is located. Despite the strong underlying support for the roles of SK1 and S1P in cancer, there is a lack in understanding of the molecular mechanisms that control SK1 activation and ultimately S1P levels. Based on these premises, we seek in Specific Aim 1 to determine the molecular mechanism of SK1 activation by APLs, both in vitro, in cells, and through structural biology, which we suggest is mediated through a novel APL binding site. Additionally, we will look at the effects of the SK1 membrane binding mutants on S1P-mediated cancer biological responses including phosphorylation of the migration and invasion associated protein Ezrin. Furthermore, I have identified novel interactions between SK1 and phosphatidylinositol phosphates (PIPs) which can also be found at the plasma membrane. I have shown that certain PIPs can de-activate SK1 even in the presence of activating APLs. This proposal, in specific Aim 2, aims at understanding these interaction and their biological consequences to cancer cell invasion and angiogenesis. Currently, there are no inhibitors of SK1 approved by the U.S. Food and Drug Administration for the treatment of any disease. Therefore, there is a need for the development of SK1 inhibitors as alternative therapeutic options for cancer. The overarching goal of these studies is to understand the molecular mechanism of SK1 activation by APLs and how PIPs can affect SK1 activity. Understanding the mechanisms by which SK1 activity is allosterically modulated will open the door to a new class of SK1 inhibitors which target SK1s ability to bind to the membrane, therefore limiting its access to its substrate. Targeting S1P production allows for a new avenue of anti-cancer therapies.

PUBLIC HEALTH RELEVANCE: Currently, cancer is the second leading cause of death in the United States. In order to develop useful therapeutics we must first understand the underlying causes of cancer development and progression. Sphingosine Kinase 1 (SK1), a lipid kinase, has been shown to be over-expressed in a number of different cancers including lung, breast, and colon among others. The product of SK1 is the bioactive sphingolipid sphingosine-1-phosphate (S1P). The cellular effects of S1P signaling include cell survival and induction of blood vessel formation, both of which contribute to the development and progression of cancer. Unfortunately, there are no FDA approved inhibitors of SK1 for the treatment of cancer. This proposal aims to characterize a novel membrane binding site which SK1 uses as a sensor for activation of its catalysis. Moreover, this proposal shows that phosphoinositol phosphates can regulate SK1 activity which is an unprecedented finding. This work will provide a proof-of-principle for targeting a unique site of SK1 to stop SK1 activation and inhibit its access to its substrate sphingosine. Furthermore, this project will identify novel SK1/lipid interactions which will link phospholipid metabolism and sphingolipid metabolism through modulation of SK1 activity.