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激活和诱导的关键靶点仍然知之甚少。这些研究将确定共同决定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癌症治疗方法。为了实现这一点，我们正在使用腺病毒感染作为一个强大但简单的遗传系统来定义p53生长调控网络，以及用于p53癌症治疗的裂解剂。腺病毒E1B-55K是病毒感染过程中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。这有望阻止病毒在正常细胞中复制，但不能在p53突变的肿瘤细胞中复制。在此前提下，Onyx-015作为一种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，但正如预期的那样，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，我们现在已经证明，即使用阿霉素、辐射或MDM2拮抗剂治疗，在感染ONYX-015的细胞中，P53激活的转录也受到抑制。使用遗传学方法，我们发现还有另一种病毒蛋白E4-ORF3，它独立于E1B-55K阻止P53的转录激活。在目标1中，我们将利用E1B-55K/E4-ORF3突变病毒作为一种强大的比较蛋白质组方法来定义关键的P53相互作用复合体，这些复合体区分(并可能确定)对阿霉素和腺病毒复制反应中P53的激活和诱导。新的P53相互作用蛋白已经被发现，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表达实验将被用来确定它们在确定P53激活和生长调节/存活中的作用。这些研究将确定关键的p53相互作用的等级，这些相互作用决定了P53在应对遗传毒性和致癌压力时的激活和抑制。在目标2中，我们将关注病毒蛋白主角E4-ORF3的上游。我们将测试假设，即E4-ORF3颠覆关键的DNA损伤信号和下游的磷酸化目标，以防止P53转录激活在Onyx-015感染细胞。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颠覆，以阻止P53靶启动子在应对遗传毒性和致癌压力时的激活。这些研究将把关键的上游信号、下游的蛋白质相互作用/修饰与P53效应物的特异性转录激活结合起来。我们将定义一个关键的新机制，使p53在腺病毒感染中失活，这将改变一个中心教条。这些新的机制洞察力将使强大的p53选择性溶瘤病毒和激活p53转录的非遗传毒性药物的合理开发成为可能。P53最早是与DNA病毒蛋白一起被发现的；这些研究将利用病毒感染来定义p53相互作用的关键网络，以及如何将它们分离用于癌症治疗。公共卫生相关性：几乎所有形式的人类癌症中的突变都会使P53肿瘤抑制因子途径失活，但目前还没有靶向药物来治疗P53突变的肿瘤细胞。P53也被腺病毒蛋白失活，我们将在这些研究中利用它来定位关键的细胞靶点，这些靶点可以被操纵来潜在地在50%的人类肿瘤中重新激活P53。此外，这项研究将提供新的机制见解，使病毒的开发成为p53突变制导导弹，并在p53突变的肿瘤细胞内特异性复制，从内到外摧毁它们。