Study of Anti-Survival signals in NF1

1型神经纤维瘤病（NF 1）是一种流行的家族性癌症综合征，影响一定数量的人类。神经纤维瘤病的异常基因位点是肿瘤抑制基因Nf 1。Nf 1（神经纤维蛋白）的蛋白质产物是一种GTP酶激活蛋白（GAP），负调节Ras信号传导，并在作为NF 1疾病细胞来源的许旺细胞中高水平表达。Nf 1的功能缺失突变或缺失导致Nf 1患者Ras通路过度激活。我们的初步数据表明，突变的Nf 1，连同蛋白激酶C（PKC）的抑制，是合成致死，通过激活有丝分裂出口检查点。在这个过程中，Plk 1以Chk 1依赖的方式被激活。Plk 1通过与cdc 14 B结合，阻断cdc 14 B的功能，进一步沉默Cdh 1的活性，导致cyclin B1稳定性增加，Nf 1缺陷型神经纤维瘤病细胞聚集于细胞周期的M期。重要的是，这种有丝分裂出口检查点的激活与细胞凋亡的诱导相一致。在酵母和哺乳动物细胞中，有丝分裂的退出涉及一系列错综复杂的事件，从分裂后期开始的姐妹染色单体分裂到胞质分裂的完成。如果损伤靶向有丝分裂的晚期阶段，则有丝分裂出口检查点将被激活，随后诱导有丝分裂灾难以消除不需要的细胞或终止有丝分裂。基于我们的初步数据和我们对有丝分裂出口检查点的了解，我们假设Nf 1突变和PKC的缺失是综合致死的，这是通过激活有丝分裂出口检查点。在这种有丝分裂危象中鉴定关键分子事件将允许神经纤维瘤病1的有效治疗干预。为了验证这一假设，我们将：1）研究Plk 1/cdc 14 B轴调节HMG处理的Nf 1缺陷型神经纤维瘤病1细胞中有丝分裂出口检查点激活的机制：2）研究Cdh 1在Nf 1缺陷型细胞中PKC抑制诱导的有丝分裂出口检查点中的作用;和3）确定Ras介导的途径如何在HMG处理的Nf 1缺陷细胞中触发有丝分裂出口检查点并进一步诱导体内细胞凋亡。由于Nf 1缺陷影响大量人群，并且晚期恶性外周神经鞘瘤（MPNST）患者的预后不良，因此迫切需要新的化疗方法来治疗这种疾病，因此这项研究的结果将在设计新的治疗策略以操纵细胞内信号传导并特异性地重新引导Nf 1缺陷细胞进行凋亡方面具有重要意义。这与NIH的中心使命完全雅阁。

DESCRIPTION (provided by applicant: Type 1 neurofibromatosis (NF1) is a prevalent familial cancer syndrome affecting certain numbers of human beings. The abnormal genetic locus in neurofibromatosis is Nf1, a tumor suppressor gene. The protein product of Nf1 (neurofibromin) is a GTPase-activating protein (GAP) that negatively regulates Ras signaling and expresses in a high level in Schwann cells that are the cell origin of NF1 disease. Loss-of- function mutations or deletion of Nf1 are responsible for hyper-activation of Ras pathway in NF1 patients. Our preliminary data demonstrated that mutated Nf1, together with the suppression of protein kinase C (PKC), is synthetically lethal, via the activation of the mitotic exit checkpoint. In this process, Plk1 was activated in a Chk1-dependent fashion. Through binding to cdc14B, Plk1 blocked its functions and further silenced Cdh1 activity, resulting in the increase of cyclin B1 stability and accumulation of Nf1 deficient neurofibromatosis cells in the M phase of the cell cycle. Importantly, the activation of this mitotic exit checkpoint coincided with the induction of apoptosis. In yeast and mammalian cells, mitotic exit involves an intricately ordered series of events, leading from the splitting of sister chromatids at anaphase onset to the completion of cell division by cytokinesis. If insults target late stages of mitosis, mitotic exit checkpoint would be activated and subsequently induce mitotic catastrophe to eliminate unwanted cells or terminates mitosis. Based on our preliminary data and our knowledge of the mitotic exit checkpoint, we hypothesize that Nf1 mutation, together with loss of PKC, is synthetically lethal, which is through the activation of the mitotic exit checkpoint. Identification of critical molecular events in this mitotic crisis will allow efficient therapeutic interventions of neurofibromatosis 1. To test this hypothesis, we will: 1) investigate the mechanism by which Plk1/cdc14B axis regulates the activation of this mitotic exit checkpoint in HMG- treated Nf1 deficient neurofibromatosis 1 cells; 2) investigate the role of Cdh1 in the mitotic exit checkpoint induced by PKC suppression in the Nf1 deficient cells; and 3) determine how Ras-mediated pathways trigger the mitotic exit checkpoint in HMG-treated Nf1 deficient cells and further induce apoptosis in vivo. Since Nf1 defect affects a large of human population and the poor prognosis for patients with advanced malignant peripheral nerve sheath tumor (MPNST) emphasize the urgent need for new chemotherapeutic approaches to treat this disease, the outcomes of this proposed study will have translational importance in the design of new therapeutic strategies to manipulate intracellular signaling and specifically re-direct the Nf1 deficient cells to undergo apoptosis, which is fully in accord with the central mission of NIH.