Defining critical p53 therapeutic targets and mechanisms

定义关键的 p53 治疗靶点和机制

• 批准号: 8433990
• 负责人: Clodagh O'Shea
• 金额: $ 35.83万
• 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/8433990
• 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 Targeting; Employee Strikes; Genetic; Genetic Transcription; Goals; Growth; Health; Human; Infection; Lytic; Malignant Neoplasms; Modification; Mutagens; 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; 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; 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.

描述（由申请人提供）：决定p53激活与诱导的关键靶点仍然知之甚少。这些研究将定义共同决定p53转录激活的相互作用的关键网络。我们的目标是操纵这些网络来开发新的合理的靶向p53癌症疗法。为了实现这一目标，我们使用腺病毒感染作为一种强大而简单的遗传系统来定义p53生长调控网络，以及用于p53癌症治疗的溶解剂。腺病毒E1 B-55 K是病毒感染中p53降解所必需的。因此，E1 B-55 K突变腺病毒ONYX-015诱导高水平的核p53。预期这可以阻止病毒在正常细胞中复制，但不能阻止p53突变肿瘤细胞中的复制。在此前提下，ONYX-015在临床试验中作为p53癌症疗法进行了测试。然而，我们发现，尽管ONYX-015诱导非常高的p53水平，正如预期的那样，p53激活的转录被抑制。这就引出了一个基本问题高水平的p53是如何失活的？为了解决这个问题，我们现在已经表明，p53激活的转录在ONYX-015感染的细胞中受到抑制，甚至在用阿霉素、辐射或Mdm 2拮抗剂处理后也是如此。使用遗传学方法，我们发现有另一种病毒蛋白E4-ORF 3，它独立于E1 B-55 K阻止p53转录激活。在目标1中，我们将利用E1 B-55 K/E4-ORF 3突变病毒的一个强大的比较蛋白质组学方法，以确定关键的p53相互作用的复合物，区分（并可能决定）激活与诱导p53在阿霉素和腺病毒复制。新的p53相互作用蛋白已经被确定，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表达实验将用于确定它们在确定p53激活和生长调节/存活中的作用。这些研究将定义决定p53激活与抑制的关键p53相互作用的层次结构，以响应遗传毒性和致癌应激。在目标2中，我们将研究上游的病毒蛋白主角E4-ORF 3。我们将测试E4-ORF 3颠覆关键DNA损伤信号和下游磷酸化靶点以防止ONYX-015感染细胞中p53转录激活的假设。在目标3中，我们将确定是否染色质修饰，转录起始或延伸被破坏，以防止p53靶启动子在响应于遗传毒性和致癌应激中的激活。这些研究将整合关键的上游信号，下游蛋白质相互作用/修饰与p53效应子的特异性转录激活。我们将定义一个关键的新机制，即p53在腺病毒感染中失活，这将改变中心法则。这些新的机制见解将使有效的p53选择性溶瘤病毒和非基因毒性药物，激活p53转录的合理发展。p53最初是与DNA病毒蛋白一起发现的;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将它们解偶联用于癌症治疗。