Polo-like kinase 1 and sphingosine-1-phosphate circuitry enhances TSC-mutant cell survival

University Of Cincinnati, Cincinnati OH

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Abstract

Renal disease (cysts, angiomyolipomas (AML), renal dysfunction, and renal cell carcinoma) is a major cause of morbidity and mortality in Tuberous Sclerosis Complex (TSC), a rare disease caused by loss-of-function mutations in TSC1 or TSC2 gene that lead to aberrant activation of the PI3K/mTOR signaling pathway. Treatment with mTORC1 inhibitors (mTORi) partially decreases the volume of angiomyolipomas in TSC, but tumors regrow when treatment is discontinued. Because of these primarily cytostatic effects of mTORi, life-long therapy is required with increased risk for side effects. Sphingolipids play key roles as mediators of cellular functions, including proliferation, necrosis, and apoptosis. Relevant to our proposal, the two central bioactive sphingolipids, ceramide and sphingosine-1-phosphate (S1P), exhibit opposing roles in regulating cell death and survival. Sphingolipid signaling is associated with mitotic events and activation of polo-like kinase 1 (PLK1). PLK1 interacts with and phosphorylates TSC1 to regulate mTORC1. TSC-deficient cells have aberrant mitosis, increased PLK1 levels and are sensitive to PLK1 inhibitors. In preliminary data we demonstrate that three key enzymes responsible for the synthesis and action of the primary bioactive sphingolipids, ceramide, and sphingosine-1-phosphate (S1P), namely dihydroceramide desaturase 1 (DEGS1), acid ceramidase (ASAH1), and sphingosine kinase 1 (SPHK1), are markedly activated in TSC2-null AML-derived cells. Our central hypothesis is that TSC mutations, by increasing the expression of PLK1 and S1P receptor 1 and 3 (S1PR1 and S1PR3), regulate the production of bioactive sphingolipids that facilitate cell survival. Successful completion of the proposed Aim 1 studies will establish stable AML-derived cells with depleted PLK1, S1PR1, and S1PR3 genes and non-targeting control using CRISPR/Cas9 gene editing technology. Successful completion of the proposed Aim 2 studies will demonstrate the biological consequences of CRISPR/Cas9 knock-out PLK1, S1PR1, and S1PR3 genes on sphingolipid actions and cell survival in vitro. The significance of this project is that it will successfully develop critical AML-derived PLK1, S1PR1, or S1PR3 gene knock-out cells with validated biological functions for future studies to investigate PLK1 and sphingolipid circuitry in TSC renal angiomyolipomas. These future studies are expected to have a positive impact because a mechanism-based understanding of sphingolipid metabolism and actions will lead to improved strategies for the treatment of TSC and other renal diseases.