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

肾脏疾病（囊肿、血管平滑肌脂肪瘤（AML）、肾功能不全和肾细胞癌）是肾硬化综合征（TSC）发病率和死亡率的主要原因，TSC是一种罕见疾病，由TSC 1或TSC 2基因功能缺失突变引起，导致PI 3 K/mTOR信号通路异常激活。用mTORC 1抑制剂（mTORi）治疗可部分减少TSC中血管平滑肌脂肪瘤的体积，但当治疗停止时，肿瘤会重新生长。由于mTORi的这些主要的细胞抑制作用，需要终身治疗，副作用的风险增加。鞘脂作为细胞功能（包括增殖、坏死和凋亡）的介质发挥关键作用。与我们的提议相关的是，两种中心生物活性鞘脂，神经酰胺和鞘氨醇-1-磷酸（S1 P），在调节细胞死亡和存活方面表现出相反的作用。鞘脂信号传导与有丝分裂事件和Polo样激酶1（PLK 1）的激活相关。PLK 1与TSC 1相互作用并磷酸化TSC 1以调节mTORC 1。TSC缺陷细胞有丝分裂异常，PLK 1水平增加，对PLK 1抑制剂敏感。在初步的数据中，我们证明，三个关键酶负责的主要生物活性鞘脂，神经酰胺，和鞘氨醇-1-磷酸（S1 P），即二氢神经酰胺去饱和酶1（DEGS 1），酸性神经酰胺酶（ASAH 1），和鞘氨醇激酶1（SPHK 1）的合成和行动，显着激活TSC 2无效AML衍生细胞。我们的中心假设是，TSC突变，通过增加PLK 1和S1 P受体1和3（S1 PR 1和S1 PR 3）的表达，调节促进细胞存活的生物活性鞘脂的产生。Aim 1研究的成功完成将使用CRISPR/Cas9基因编辑技术建立具有耗尽的PLK 1，S1 PR 1和S1 PR 3基因和非靶向对照的稳定AML衍生细胞。目标2研究的成功完成将证明CRISPR/Cas9敲除PLK 1、S1 PR 1和S1 PR 3基因对鞘脂作用和细胞存活的生物学后果。该项目的意义在于，它将成功开发具有验证生物学功能的关键AML衍生PLK 1，S1 PR 1或S1 PR 3基因敲除细胞，用于未来研究TSC肾血管平滑肌脂肪瘤中的PLK 1和鞘脂回路。这些未来的研究预计将产生积极的影响，因为对鞘脂代谢和作用的机制的理解将导致改善TSC和其他肾脏疾病的治疗策略。

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.