Defining critical p53 therapeutic targets and mechanisms

定义关键的 p53 治疗靶点和机制

• 批准号: 8212429
• 负责人: Clodagh O'Shea
• 金额: $ 38.12万
• 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/8212429
• 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; 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. 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）：决定 p53 激活与诱导的关键靶点仍知之甚少。这些研究将定义共同决定 p53 转录激活的关键相互作用网络。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但简单的遗传系统来定义 p53 生长调节网络，以及 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 突变腺病毒 ONYX-015 诱导高水平的核 p53。预计这会阻止正常细胞中的病毒复制，但不会阻止 p53 突变的肿瘤细胞中的病毒复制。在此前提下，ONYX-015作为p53癌症疗法在临床试验中进行了测试。然而，我们发现，尽管ONYX-015诱导了非常高的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，我们现已证明，即使在使用阿霉素、辐射或 Mdm2 拮抗剂治疗后，ONYX-015 感染的细胞中 p53 激活的转录也会受到抑制。通过基因方法，我们发现还有另一种病毒蛋白 E4-ORF3，它可以独立于 E1B-55K 阻止 p53 转录激活。在目标 1 中，我们将利用 E1B-55K/E4-ORF3 突变病毒进行强大的比较蛋白质组学方法，以定义关键的 p53 相互作用复合物，该复合物区分（并可能确定）响应阿霉素和腺病毒复制的 p53 激活与诱导。新型 p53 相互作用蛋白已被鉴定，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。这些研究将定义关键 p53 相互作用的层次结构，决定 p53 响应基因毒性和致癌应激的激活与抑制。在目标 2 中，我们将向上游寻找病毒主角蛋白 E4-ORF3。我们将测试以下假设：E4-ORF3 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。在目标 3 中，我们将确定染色质修饰、转录起始或延伸是否被破坏，以防止响应基因毒性和致癌应激而激活 p53 靶启动子。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 效应子的特定转录激活。我们将定义一种关键的新机制，使 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 选择性溶瘤病毒和激活 p53 转录的非基因毒性药物的合理开发成为可能。 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开以进行癌症治疗。公共健康相关性：p53 肿瘤抑制途径因几乎所有形式的人类癌症中的突变而失活，但尚无治疗 p53 突变肿瘤细胞的靶向药物。 p53 也会被腺病毒蛋白灭活，我们将在这些研究中利用腺病毒蛋白来查明关键的细胞靶标，这些靶标可被操纵以潜在地“重新激活”50% 人类肿瘤中的 p53。此外，这项研究将提供新的机制见解，使开发出充当 p53 突变制导导弹的病毒成为可能，并在 p53 突变肿瘤细胞内特异性复制，使它们从内到外内爆。