Selective Targeting of MEK and AKt in Lymphoma and Myeloma Apoptosis

描述(申请人提供)：美国每年新增癌症病例超过100万例，癌症死亡人数每年超过50万人。在肿瘤中获得耐药性是癌症治疗中一个持续存在的问题。因此，我们的广泛目标是了解赋予肿瘤细胞抗凋亡能力的生物信号，重点是淋巴瘤。大约三分之一的肿瘤含有功能失调的RAS。因此，我们最近开始研究一种天然的抗癌药物Manumycin-A的作用机制。该化合物是一种法尼基转移酶抑制剂(FTI)，是一系列阻断RAS法尼化的药物。法尼化是RAS成熟和插入质膜所必需的翻译后过程，对正常和功能失调的RAS的最终激活都是必不可少的。我们发现，无论RAS效应器的激活状态如何，Man-A都能杀死一组淋巴和髓系肿瘤。在所有肿瘤中，Man-A诱导的ROS位于caspase激活的上游。其次是选择性的、ROS和caspase依赖的MEK和Akt的切割。我们还发现了强有力的证据，表明丝氨酸/苏氨酸蛋白磷酸酶1(PP1)是Man-A介导的死亡反应的初始效应器。因此，PP1的取消阻断了Man-A的所有下游效应，并导致MEK和Akt的稳定。MEK和Akt磷酸化的稳定阻止了它们的切割，从而使原本易感的肿瘤对诱导凋亡死亡具有抵抗力。因此，PP1似乎是死亡级联中的目标或近端参与者。因此，我们的目标是结合生物化学、分子和蛋白质组学的方法，在MEK和Akt的启动和靶向蛋白水解性切割中确定ROS和蛋白磷酸酶活性的相互关系。我们的具体目标是：1)建立对Man-A及其相互作用的生化反应的良好动力学和相互依赖；2)利用分子方法确定PP1在FTI介导的细胞凋亡中的作用；以及3)利用蛋白质组学确定MEK和Akt上的裂解位点，并确定氧化在后续加工中的作用。这些研究最终将有助于靶向这些途径，使肿瘤重新敏感，从而达到治疗消除的目的。

DESCRIPTION (provided by applicant): There are greater than 1,000,000 new cancer cases per year in the United States and cancer deaths exceed 500,000 annually. The acquisition of drug resistance in tumors is an ongoing problem in cancer therapy. Therefore, our broad aim is to understand the biological signals that confer resistance to apoptosis on tumor cells, with a focus on lymphomas. About one third of all tumors harbor dysfunctional Ras. Therefore, we recently began studies to understand the mechanism of action of a natural tumoricide, Manumycin-A. This compound, is a farnesyltransferase inhibitor (FTI), a family of agents that blocks Ras farnesylation. Farnesylation is a posttranslational process that is required for Ras maturation and insertion into the plasma membrane and is essential for ultimate activation of both normal and dysfunctional Ras. We found that Man-A killed a panel of lymphoid and myeloid tumors irrespective of the activation state of Ras effectors. In all the tumors, Man-A induced reactive oxygen species (ROS) which were upstream of caspase activation. This was followed by selective, ROS and caspase-dependent cleavage of MEK and Akt. We also found strong evidence suggesting a role of the serine/threonine protein phosphatase 1 (PP1) as an initial effector of the Man-A mediated death response. Thus, abrogation of PP1 blocked all downstream effects of Man-A and resulted in stabilization of MEK and Akt. Stabilization of MEK and Akt phosphorylation prevented their cleavage and thus, conferred resistance to the induction of apoptotic death on the otherwise susceptible tumors. Hence, PP1 appears to be a target or proximal player in the death cascade. Therefore, our goals are to determine the interrelationships of ROS, and protein phosphatase activities in the priming and targeting of MEK and Akt for proteolytic cleavage using a combination of biochemical, molecular and proteomic approaches. Our specific aims are to: 1) establish fine kinetics and interdependence of biochemical responses to Man-A and each other; 2) establish the role of PP1 in FTI-mediated apoptosis using molecular approaches; and 3) to determine the cleavage sites on MEK and Akt and determine the role of oxidation in subsequent processing, using proteomics. These studies will ultimately help in the targeting of these pathways to re-sensitize tumors for therapeutic elimination.