Defining critical p53 therapeutic targets and mechanisms

定义关键的 p53 治疗靶点和机制

• 批准号: 7567632
• 负责人: Clodagh O'Shea
• 金额: $ 39.3万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7567632
• 关键词: Address; Adenovirus Infections; Adenoviruses; Adriamycin PFS; Binding; Carcinogens; Cell physiology; Cells; Chromatin; Clinical Trials; Complex; DNA; DNA Damage; DNA damage checkpoint; Data; Development; Drug Delivery Systems; Employee Strikes; Genetic; Genetic Transcription; Goals; Growth; Human; Infection; Lytic; Malignant Neoplasms; Modification; Mutation; Normal Cell; Nuclear; ONYX-015; Oncogenic; Oncolytic viruses; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Protein p53; Proteins; Proteomics; RNA Interference; Regulation; Repression; Research; Role; Signal Transduction; Stress; System; TP53 gene; Testing; Transcriptional Activation; Viral; Viral Proteins; Virus; Virus Diseases; cDNA Expression; cancer therapy; cellular targeting; chemotherapy; chromatin immunoprecipitation; chromatin modification; comparative; design; genotoxicity; histone modification; insight; interest; irradiation; kinase inhibitor; mutant; neoplastic cell; novel; prevent; promoter; protein protein interaction; public health relevance; research study; response; therapeutic target; tumor; virus development

DESCRIPTION (provided by applicant): The key targets that determine the activation versus the induction of p53 are still poorly understood. These studies will define the critical networks of interactions that together determine p53 transcriptional activation. Our objective is to manipulate these networks to develop new rational targeted p53 cancer therapies. To achieve this we are using adenovirus infection as a powerful but simple genetic system to define the p53 growth regulatory network, as well as a lytic agent for p53 cancer therapy. Adenovirus E1B-55K is required for p53 degradation in viral infection. Thus, an E1B-55K mutant adenovirus, ONYX-015, induces high levels of nuclear p53. This was expected to prevent viral replication in normal cells, but not in p53-mutant tumor cells. On this premise, ONYX-015 was tested in clinical trials as a p53 cancer therapy. However, we discovered that although ONYX-015 induces very high p53 levels, as expected, p53 activated transcription was suppressed. This leads to a fundamental question-how are high levels of p53 inactivated? To address this, we have now shown that p53 activated transcription is suppressed in ONYX-015 infected cells, even upon treatment with adriamycin, irradiation or Mdm2 antagonists. Using a genetic approach, we have discovered that there is another viral protein, E4-ORF3, which prevents p53 transcriptional activation independently of E1B-55K. In Aim 1 we will exploit E1B-55K/E4-ORF3 mutant viruses for a powerful comparative proteomics approach to define critical p53 interacting complexes that distinguish (and likely determine) activation versus induction of p53 in response to adriamycin and adenoviral replication. Novel p53-interacting proteins have already been identified and our preliminary data indicate that they are critical determinants of p53 activation. RNAi and cDNA expression experiments will be used to define their role in determining p53 activation and growth regulation/survival. These studies will define the hierarchy of critical p53 interactions that determine p53 activation versus repression in response to genotoxic and oncogenic stress. In Aim 2, we will look upstream to the viral protein protagonist, E4-ORF3. We will test the hypothesis that E4-ORF3 subverts key DNA damage signals and downstream phosphorylation targets to prevent p53 transcriptional activation in ONYX-015 infected cells. In Aim 3 we will determine if chromatin modifications, transcriptional initiation or elongation are subverted to prevent the activation of p53 target promoters in reponse to genotoxic and oncogenic stress. These studies will integrate the critical upstream signals, downstream protein interactions/modifications with the specific transcriptional activation of p53 effectors. We will define a critical new mechanism whereby p53 is inactivated in adenovirus infection, which will change a central dogma. These new mechanistic insights will enable the rational development of potent p53 selective oncolytic viruses and non-genotoxic drugs that activate p53 transcription. p53 was first discovered with a DNA viral protein; these studies will exploit viral infection to define the critical networks of p53 interactions and how to uncouple them for cancer therapy. PUBLIC HEALTH RELEVANCE: The p53 tumor suppressor pathway is inactivated by mutations in almost every form of human cancer, but there are no targeted drugs to treat p53 mutant tumor cells. p53 is also inactivated by adenoviral proteins, which we will exploit in these studies to pinpoint critical cellular targets that could be manipulated to potentially `re-activate' p53 in 50% of human tumors. In addition, this research will provide new mechanistic insights that will enable the development of viruses that act as p53-mutation guided missiles, and which specifically replicate within p53 mutant tumor cells to implode them from the inside out.

描述（由申请人提供）：确定激活与p53诱导的关键目标仍然很少了解。这些研究将定义相互作用的关键网络，这些网络共同确定p53转录激活。我们的目标是操纵这些网络，以开发新的合理靶向p53癌症疗法。为了实现这一目标，我们将腺病毒感染用作强大但简单的遗传系统来定义p53生长调节网络，以及用于p53癌症治疗的裂解剂。腺病毒E1B-55K是病毒感染中p53降解所必需的。因此，E1B-55K突变体腺病毒Onyx-015诱导了高水平的核p53。预计这将防止正常细胞中的病毒复制，但不能在p53突变的肿瘤细胞中复制。在此前提下，Onyx-015在临床试验中作为p53癌症治疗进行了测试。但是，我们发现，尽管Onyx-015诱导了非常高的p53水平，但如预期的那样，p53激活的转录被抑制了。这导致了一个基本的问题 - 高水平的p53灭活了吗？为了解决这个问题，我们现在已经表明，即使用adriamycin，iradiation或MDM2拮抗剂治疗，p53激活的转录也会在Onyx-015感染的细胞中受到抑制。使用遗传方法，我们发现还有另一种病毒蛋白E4-ORF3，它可以独立于E1B-55K来防止p53转录激活。在AIM 1中，我们将利用E1B-55K/E4-ORF3突变病毒来使用强大的比较蛋白质组学方法来定义关键的p53相互作用的复合物，从而区分（并可能确定）激活p53的激活与p53的响应响应于adriamycin和adenenoviral重复。新型的p53相互作用蛋白已经被鉴定出来，我们的初步数据表明它们是p53激活的关键决定因素。 RNAi和cDNA表达实验将用于定义其在确定p53激活和生长调节/存活中的作用。这些研究将定义临界p53相互作用的层次结构，这些相互作用确定p53激活与抑制遗传毒性和致癌应激。在AIM 2中，我们将在病毒蛋白主角E4-ORF3上游查找。我们将检验以下假设：E4-ORF3颠覆了关键的DNA损伤信号和下游磷酸化靶标，以防止Onyx-015受感染细胞中的p53转录激活。在AIM 3中，我们将确定染色质修饰，转录启动或伸长是否被颠覆以防止p53靶启动子激活基因毒性和致癌应激。这些研究将将关键的上游信号，下游蛋白质相互作用/修饰与p53效应子的特定转录激活相结合。我们将定义一种关键的新机制，在该机制中p53在腺病毒感染中被灭活，这将改变中心的教条。这些新的机械见解将使有效的p53选择性肿瘤病毒和非生物毒性药物的合理发展，这些药物激活p53转录。 p53首先是用DNA病毒蛋白发现的。这些研究将利用病毒感染来定义p53相互作用的关键网络，以及如何使它们脱离癌症治疗。公共卫生相关性：p53肿瘤抑制途径几乎每种形式的人类癌症都被突变灭活，但是没有针对治疗p53突变肿瘤细胞的靶向药物。 p53也被腺病毒​​蛋白灭活，我们将在这些研究中利用这些蛋白质来查明可以操纵的关键细胞靶标，这些靶标可能会在50％的人类肿瘤中操纵以潜在地“重新激活” p53。此外，这项研究将提供新的机械见解，以使其能够发育为P53 Monted导弹的病毒，并在p53突变肿瘤细胞中特别复制以从内而外爆炸。