Defining critical p53 therapeutic targets and mechanisms

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癌症疗法。为了实现这一目标，我们正在使用腺病毒感染作为一个强大而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。在病毒感染中，腺病毒E1B-55K是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的复制，但在p53突变的肿瘤细胞中却不能。在此前提下，ONYX-015作为p53癌症治疗药物进行了临床试验。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p53激活的转录在ONYX-015感染的细胞中被抑制，即使用阿霉素、辐射或Mdm2拮抗剂治疗。利用遗传方法，我们发现了另一种病毒蛋白E4-ORF3，它可以独立于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，这些复合物在阿霉素和腺病毒复制的反应中区分（并可能决定）p53的激活与诱导。新的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表达实验将用于确定它们在决定p53激活和生长调节/生存中的作用。这些研究将确定关键p53相互作用的层次结构，这些相互作用决定了p53在基因毒性和致癌压力下的激活与抑制。在目标2中，我们将着眼于上游的病毒蛋白主角E4-ORF3。我们将在ONYX-015感染细胞中验证E4-ORF3破坏关键DNA损伤信号和下游磷酸化靶点以阻止p53转录激活的假设。在Aim 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p53靶启动子在基因毒性和致癌应激下的激活。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效应子的特定转录激活。我们将定义p53在腺病毒感染中失活的关键新机制，这将改变中心教条。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病毒蛋白一起被发现的；这些研究将利用病毒感染来确定p53相互作用的关键网络，以及如何解耦它们以进行癌症治疗。

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.