Project Summary/Abstract Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia and accounts for 20% of leukemia-related deaths annually. In CLL, hyperactivation of the B-cell receptor (BCR) response leads to over- activation of many downstream effectors that drive proliferation and survival, including Bruton’s Tyrosine Kinase (BTK), LYN Kinase and the Signal Transducer and Activator of Transcription 3 (STAT3). This, along with interactions with the microenvironment, leads to metabolic reprogramming and increased metabolic plasticity, allowing CLL cells to thrive under various conditions. Our lab has previously shown that delivery of ceramide, via a ceramide nanoliposome (CNL), targets this metabolic plasticity and disrupts the STAT3 signaling pathway, leading to decreased transcriptional activity, impaired glycolysis and cell death in multiple CLL models, including patient samples. However, restoration of glycolysis and/or STAT3 transcriptional activity only partially protects from CNL-induced cell death, suggesting that other mechanisms are at play. Here, using an advanced mitochondrial metabolomics workflow, I show that CNL disrupts oxidative phosphorylation (OXPHOS) by selectively inhibiting Complex III of the electron transport chain. This disruption of OXPHOS is time dependent and correlates with a CNL induced upregulation of glycosphingolipids in both whole cells and mitochondrial extracts, suggesting an unexpected link. Importantly, while glycosphingolipids are typically seen as a protective shunt away from more apoptotic ceramides, I have shown that inhibition of glucosylceramide (the gateway lipid to the glycosphingolipid pathway) formation protects from CNL mediated mitochondrial dysfunction and cell death in CLL, further supporting this connection. Lastly, glucosylceramide blockade also diminishes the ability of CNL to alter LYN and BTK phosphorylation. Given these effects, I hypothesize that sialic acid containing glycosphingolipids downstream of glucosylceramide activate SIGLECS (the sialic acid binding proteins known to negatively regulate BCR signaling) leading to mitochondrial dysfunction and cell death. Insights into this area will provide a novel therapeutic framework for CLL patients and deepen our understanding of lipid/protein biology. The first aim of this proposal (F99 Phase) identifies the key glycosphingolipid metabolites driving these effects, determines the mechanism by which they alter BCR signaling effectors and mitochondrial function, and evaluates their therapeutic potential in vivo. The knowledge and skills learned from these studies will then be used to study the role of lipid metabolism in the metabolic plasticity of glioma stem cells and in the mechanisms governing mitochondrial transfer between these cells and astrocytes in the context of glioblastoma (K99 Phase).