Selective Targeting of MEK and AKt in Lymphoma and Myeloma Apoptosis

描述（由申请人提供）：美国每年有超过1，000，000例新发癌症病例，每年癌症死亡人数超过500，000人。肿瘤耐药性的获得是癌症治疗中的一个持续问题。因此，我们的广泛目标是了解赋予肿瘤细胞凋亡抗性的生物信号，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，我们最近开始研究了解天然杀肿瘤剂Manumycin-A的作用机制。这种化合物是一种法尼基转移酶抑制剂（FTI），是一种阻断Ras法尼基化的药物家族。法尼基化是Ras成熟和插入质膜所需的翻译后过程，并且对于正常和功能失调的Ras的最终激活是必不可少的。我们发现，Man-A杀死了一组淋巴和骨髓肿瘤，而不管Ras效应子的激活状态如何。在所有的肿瘤中，Man-A诱导活性氧（ROS），这是caspase激活的上游。随后是MEK和Akt的选择性、ROS和半胱天冬酶依赖性切割。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（PP 1）作为Man-A介导的死亡反应的初始效应物的作用。因此，PP 1的废除阻断了Man-A的所有下游效应，并导致MEK和Akt的稳定。MEK和Akt磷酸化的稳定化阻止了它们的切割，因此赋予了对在其他方面易感的肿瘤上诱导凋亡性死亡的抗性。因此，PP 1似乎是死亡级联中的目标或近端参与者。因此，我们的目标是使用生物化学、分子和蛋白质组学方法的组合来确定ROS和蛋白磷酸酶活性在MEK和Akt的引发和靶向蛋白水解裂解中的相互关系。我们的具体目标是：1）建立对Man-A和彼此的生化反应的精细动力学和相互依赖性; 2）使用分子方法建立PP 1在FTIR介导的细胞凋亡中的作用;和3）使用蛋白质组学确定MEK和Akt上的切割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，以进行治疗消除。

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.