Survival Mechanisms in Leukemic NK Cells

描述（由申请人提供）：该项目的长期目标是开发更好的NK-LGL白血病治疗方法。该建议通过假设鞘脂变阻器的不平衡赋予白血病表型，代表了白血病发病机制的新方法。该模型假设细胞命运是由促生存（鞘氨醇-1-磷酸，S1P）和促凋亡（神经酰胺）脂质之间的平衡决定的。初步数据表明，这种疾病的鞘脂代谢改变，包括白血病LGL中S1P水平升高和神经酰胺水平降低；酸性神经酰胺酶表达增加；并且发现酸性神经酰胺酶的稳定敲除促进长链神经酰胺的产生，并导致白血病LGL的生存能力下降。此外，我们表明靶向鞘脂信号代表了治疗干预这种疾病的新途径。在Fischer大鼠动物模型中，我们利用纳米脂质体制剂的短链C6神经酰胺或S1P受体拮抗剂FTY720实现了侵袭性NK-LGL白血病的完全缓解。我们将验证S1PR5信号通路介导白血病NK细胞存活的假设（Specific Aim 1）。在白血病NK细胞上表达的主要S1P受体S1PR5的下调抑制了ERK的组成性磷酸化并诱导细胞凋亡。在白血病NK细胞中观察到S1PR5的潜在下游效应物Rac1、PAK3、RhoB和ROCK1的组成性过表达。药理抑制Gi、RAC1和ROCK也会导致白血病NK细胞死亡，表明这些靶点可能参与s1pr5依赖的生存途径。特异性目的2将验证FTY720通过靶向鞘脂信号诱导白血病NK细胞凋亡和自噬的假设。我们发现，药物抑制自噬可增加FTY720诱导的细胞凋亡，表明阻断自噬可增强FTY720对白血病NK细胞的治疗效果。FTY720治疗白血病LGL导致细胞内S1P积累，提示FTY720可能通过抑制S1P裂解酶诱导自噬发生。此外，我们假设白血病NK细胞依赖于NK受体靶识别在体内产生的生存信号。令人信服的初步数据表明，正常NK细胞在体外靶结合后，鞘脂存活途径被激活。与NK-LGL白血病相似的发现包括S1PR5上调，RhoB、ROCK1、Rac1和PAK3被鉴定为S1PR5信号的下游组分，FTY720诱导细胞凋亡。我们预计本研究将对NK细胞生物学领域产生重大影响。我们的工作将定义白血病和活化正常NK细胞生存的重要新途径。我们期望理解鞘脂存活信号将为无法治愈的疾病带来新的治疗方法。

DESCRIPTION (provided by applicant): The long term goal of this project is to develop better therapeutics for NK-LGL leukemia. The proposal represents a new approach to leukemia pathogenesis by positing that imbalances in the sphingolipid rheostat confer the leukemia phenotype. This model postulates that cell fate is determined by the balance between pro- survival (sphingosine-1-phosphate, S1P) and pro-apoptotic (ceramide) lipids. Preliminary data demonstrate altered sphingolipid metabolism in this disease, including increased levels of S1P and decreased levels of ceramide in leukemic LGL; increased expression of acid ceramidase; and findings that stable knockdown of acid ceramidase enhances production of long chain ceramide and results in decreased viability of leukemic LGL. Furthermore, we show that targeting sphingolipid signaling represents a new avenue for therapeutic intervention in this disease. We achieve complete remission of aggressive NK-LGL leukemia by utilizing nanoliposomal formulation of short chain C6 ceramide or the S1P receptor antagonist, FTY720, in the Fischer rat animal model. We will test the hypothesis that S1PR5 signaling mediates survival of leukemic NK cells (Specific Aim 1). Knockdown of S1PR5, the predominant S1P receptor expressed on leukemic NK cells, inhibits constitutive ERK phosphorylation and induces apoptosis. Constitutive overexpression of Rac1, PAK3, RhoB and ROCK1, potential downstream effectors of S1PR5, is observed in leukemic NK cells. Pharmacological inhibition of Gi, RAC1 and ROCK also results in cell death in leukemic NK cells, demonstrating that these targets may be involved in S1PR5-dependent survival pathways. Specific Aim 2 will test the hypothesis that FTY720 induces both apoptosis and autophagy in leukemic NK cells by targeting sphingolipid signaling. We show that pharmacological inhibition of autophagy increases apoptosis induced by FTY720, indicating that blockade of autophagy potentiates FTY720 therapeutic efficacy in leukemic NK cells. FTY720 treatment of leukemic LGL leads to accumulation of intracellular S1P, suggesting that autophagy induced by FTY720 might occur through inhibition of S1P lyase. In addition, we postulate that leukemic NK cells are dependent on survival signaling resulting from NK receptor target recognition in vivo. Convincing preliminary data indicate that sphingolipid survival pathways are activated in normal NK cells after target binding in vitro. Findings similar to those seen in NK-LGL leukemia include upregulation of S1PR5, identification of RhoB, ROCK1, Rac1 and PAK3 as downstream components of S1PR5 signaling, and induction of apoptosis by FTY720. We anticipate that the proposed research will have significant impact on the field of NK cell biology. Our work will define new pathways important for survival of both leukemic and activated normal NK cells. We expect that understanding sphingolipid survival signaling will lead to novel therapeutic approaches for an incurable illness.