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）、肾功能障碍和肾细胞癌）是结节性硬化症（TSC）发病和死亡的主要原因，TSC是一种罕见的疾病，由TSC1或TSC2基因的功能丧失突变引起PI3K/mTOR信号通路的异常激活。使用mTORC1抑制剂（mTORi）治疗可部分减少TSC中血管平滑肌脂肪瘤的体积，但当停止治疗时，肿瘤会重新生长。由于这些主要的细胞抑制作用，mTORi需要终身治疗，副作用的风险增加。鞘脂作为细胞功能的介质发挥关键作用，包括增殖、坏死和凋亡。与我们的建议相关，神经酰胺和鞘氨醇-1-磷酸（S1P）这两种中枢生物活性鞘脂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 （PLK1）的激活有关。PLK1与TSC1相互作用并磷酸化以调节mTORC1。tsc缺陷细胞有丝分裂异常，PLK1水平升高，对PLK1抑制剂敏感。在初步数据中，我们证明了三个关键酶，即二氢神经酰胺去饱和酶1 (DEGS1)，酸性神经酰胺酶（ASAH1）和鞘氨醇激酶1 (SPHK1)，在tsc2缺失的aml衍生细胞中显着激活。我们的中心假设是TSC突变通过增加PLK1和S1P受体1和3 （S1PR1和S1PR3）的表达，调节促进细胞存活的生物活性鞘脂的产生。成功完成拟议的Aim 1研究将使用CRISPR/Cas9基因编辑技术建立稳定的aml来源细胞，其缺失PLK1， S1PR1和S1PR3基因和非靶向控制。成功完成拟议的Aim 2研究将证明CRISPR/Cas9敲除PLK1、S1PR1和S1PR3基因对鞘脂作用和体外细胞存活的生物学影响。该项目的意义在于，它将成功开发出具有生物学功能的aml来源的关键PLK1、S1PR1或S1PR3基因敲除细胞，为未来研究TSC肾血管平滑肌脂肪瘤中的PLK1和鞘脂通路提供基础。这些未来的研究有望产生积极的影响，因为基于机制的鞘脂代谢和作用的理解将导致改善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.