Exploring the interaction of lipid metabolism with the hexosamine pathway and tumor growth

PROJECT SUMMARY Cancer metabolic reprogramming is key for the adaptation of the tumors to their harsh microenvironments. Elevated LD abundance in tumors is frequently detected and is being recognized as a common feature of their rewired metabolism. Lipid droplets are multifaceted organelles that participate in crucial cellular functions including metabolism, membrane synthesis and homeostasis, signaling, and protein trafficking. We and others have shown that hypoxia stimulates LD accumulation and that forced depletion of LD stores impedes tumor growth. In order to discover novel regulators of lipid droplet turnover, we performed a genome-wide loss of function screen based on Crispr engineering. Edited cells were separated based on their LD abundance and sequenced. Our approach identified GFPT1 (glutamine-fructose-6-phosphate transaminase 1) as a candidate positive LD regulator. GFPT1 catalyzes the first and rate limiting step in hexosamine biosynthesis. The hexosamine biosynthetic pathway uses a glycolytic intermediate and glutamine to produce UDP-GlcNAc which is essential for O-GlcNACylation and glycosylation of proteins and other macromolecules. Flux through the hexosamine pathway is regulated by oncogenic events, nutrient availability, and input from nutrient sensors like AMPK. In turn, HBP impacts crucial signaling functions and metabolic processes. Cancers have elevated HBP activity and gene expression and are dependent on this activity for survival, growth, and metastatic dissemination. Broad range pharmacological inhibitors of the HBP have shown promising results as a therapeutic approach, however, there are no HBP-directed cancer therapies currently approved. Our preliminary finding that GFPT1 positively regulates lipid storage prompts us to hypothesize that concurrent inhibition of the HBP and neutral lipid synthesis will result in more complete LD depletion and enhanced tumor control. In aim 1 we will delineate the lipid metabolic processes impacted by genetic GFPT1 ablation or small molecule inhibition of HBP in PDAC models in vitro. We will uncover nutritional requirements for LD turnover under normoxia and hypoxia in the context of HBP inhibition and will quantify the toxicity of combined HBP- and lipid metabolism inhibition. Further, we will mechanistically link GFPT1 and AMPK via post-translational modifications during normoxic and hypoxic LD accumulation. In aim 2 we will evaluate the in vivo efficacy of HBP and metabolic inhibitors in orthotopic PDAC models and will measure the impact of these new treatment schemes on tumor microenvironmental characteristics. Successful completion of this proposal will uncover a new connection between two cancer-promoting metabolic programs and will develop new chemotherapeutic paradigms for cancer treatment.