Survival Mechanisms in Leukemic NK Cells

描述(申请人提供)：这个项目的长期目标是开发更好的治疗NK-LGL白血病的方法。这项提议提出了一种新的白血病发病机制的方法，即鞘磷脂变阻器的失衡导致了白血病的表型。这个模型假设细胞的命运是由促进生存的(鞘氨醇-1-磷酸，S1P)和促进凋亡的(神经酰胺)脂之间的平衡决定的。初步数据显示，在这种疾病中鞘磷脂代谢改变，包括白血病LGL中S1P水平增加和神经酰胺水平下降；酸性神经酰胺酶表达增加；以及发现稳定抑制酸性神经酰胺酶增加了长链神经酰胺的产生，导致白血病LGL存活率下降。此外，我们还表明，靶向鞘脂信号转导为治疗该疾病提供了一条新途径。我们利用短链C6神经酰胺或S1P受体拮抗剂FTY720的纳米脂质体制剂，在Fischer大鼠模型中实现了侵袭性NK-LGL白血病的完全缓解。我们将验证S1PR5信号介导白血病NK细胞存活的假设(特定目标1)。S1PR5是白血病NK细胞上主要表达的S1P受体，S1PR5的敲除可抑制ERK的结构性磷酸化，诱导细胞凋亡。在白血病NK细胞中观察到S1PR5的潜在下游效应分子rac1、PAK3、RhoB和ROCK1的结构性过表达。GI、RAC1和ROCK的药理抑制也导致白血病NK细胞死亡，表明这些靶点可能参与S1PR5依赖的生存通路。特定目的2将验证FTY720通过靶向鞘脂信号而诱导白血病NK细胞凋亡和自噬的假设。我们发现，药物抑制自噬增加了FTY720诱导的细胞凋亡，表明阻断自噬增强了FTY720对白血病NK细胞的治疗效果。FTY720处理白血病LGL后导致细胞内S1P积聚，提示FTY720诱导的自噬可能是通过抑制S1P裂解酶而发生的。此外，我们假设白血病NK细胞依赖于体内NK受体靶点识别所产生的生存信号。令人信服的初步数据表明，在体外靶点结合后，正常NK细胞中的鞘磷脂生存通路被激活。与NK-LGL白血病相似的发现包括S1PR5上调，发现RhoB、ROCK1、rac1和PAK3是S1PR5信号的下游成分，以及FTY720诱导细胞凋亡。我们预计，这项拟议的研究将对NK细胞生物学领域产生重大影响。我们的工作将定义对白血病和激活的正常NK细胞的生存至关重要的新途径。我们希望了解鞘磷脂生存信号将导致对这种不治之症的新的治疗方法。

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.