Defining Critical p53 Therapeutic Targets and Mechanisms

描述（由申请人提供）：本次更新的目标是探索灭活p53的病毒机制的新见解，以开发有效的p53肿瘤选择性溶瘤腺病毒疗法。p53通路在几乎所有的人类癌症中都会因突变而失活。然而，尽管如此，仍然没有批准的针对p53缺陷肿瘤细胞的合理治疗方法。腺病毒E1B-55K以p53为目标进行降解，这被认为是p53失活和病毒复制的必要条件。在这个前提下，一种B-55K病毒ONYX-015在临床试验中作为p53癌症疗法进行了测试。然而，患者的反应与肿瘤的p53状态无关。虽然p53是诱导的，但它是无活性的。因此，p53不能决定ONYX-015的肿瘤选择性复制。因此，如果确定腺病毒灭活p53的E1B-55K独立机制，p53选择性的巨大潜力仍然可以实现。这是上一个赠款期的目标。这导致发现Ad5编码另一种蛋白E4-ORF3，该蛋白通过形成一种核聚合物使p53靶启动子从头异染色质沉默，从而阻止p53- dna结合，从而使p53失活。由于无法进入，p53无法激活下游效应物的转录来限制病毒复制。这些研究改变了长期以来关于p53在腺病毒感染中如何失活的定义。在这里，我们建议利用这些机制的见解来设计E1B-55K/E4-ORF3突变腺病毒，作为p53选择性溶瘤病毒治疗的令人兴奋的新药物。这需要E1B-55K和E4-ORF3突变选择性地将p53失活与其在病毒复制中的其他功能分离。先前的研究显示E1B-55K h60a突变符合这些标准。为了确定E4-ORF3的类似突变，我们求解了Ad5 E4-ORF3的原子结构。在Aim 1中，我们将利用这一点揭示沉默p53靶基因的新型结构基序。我们已经发现来自其他腺病毒亚群的E4-ORF3蛋白不能灭活p53。这为鉴定沉默p53靶基因的Ad5 E4-ORF3特异性基序和设计具有E4-ORF3离散突变阻止p53失活的病毒提供了合理的基础。在Aim 2中，我们将结合E4ORF3和E1B-55K突变来开发新的Ad5和Ad34病毒，并在正常和肿瘤细胞面板上测试它们的p53选择性。这些研究将确定p53阻止病毒复制的关键功能，以及它们是否被癌症中的p53和p14ARF突变破坏。最后，在Aim 3中，我们将在临床前小鼠癌症模型中测试新型p53选择性溶瘤病毒。我们将利用新技术来设计额外的基因组修饰，以防止病毒在肝脏中的摄取和限制炎症，从而实现全身传递和扩大的肿瘤趋向性。这些病毒疗法具有自我延续的潜力，通过调节细胞死亡来杀死肿瘤，并且

DESCRIPTION (provided by applicant): The goal of this renewal is to exploit new insights into viral mechanisms that inactivate p53 to develop potent p53 tumor selective oncolytic adenovirus therapies. The p53 pathway is inactivated by mutations in almost every form of human cancer. However, despite this, there are still no approved rational therapies that target p53 defective tumor cells. Adenovirus E1B-55K targets p53 for degradation, which was thought to be essential for p53 inactivation and viral replication. On this premise, a �B-55K virus, ONYX-015, was tested in clinical trials as a p53 cancer therapy. However, patient responses did not correlate with the p53 status of their tumors. Although p53 is induced, it is inactive. Consequently, p53 does not determine the tumor selective replication of ONYX-015. As such, the enormous potential of a p53 selective could still be realized if the E1B-55K independent mechanisms through which adenovirus inactivates p53 are determined. This was the goal of the previous grant period. This led to the discovery that Ad5 encodes another protein, E4-ORF3, which inactivates p53 by forming a nuclear polymer that specifies de novo heterochromatin silencing of p53 target promoters, thereby preventing p53-DNA binding. With access denied, p53 is powerless to activate the transcription of downstream effectors to limit viral replication. These studies changed the longstanding definition of how p53 is inactivated in adenovirus infection. Here we propose to exploit these mechanistic insights to engineer E1B-55K/E4-ORF3 mutant adenoviruses as exciting new agents for p53 selective oncolytic viral therapy. This requires E1B-55K and E4-ORF3 mutations that selectively uncouple p53 inactivation from their other functions in viral replication. Previous studies revealed an E1B-55K H260A mutation that fulfills these criteria. To identify analogous mutations in E4-ORF3, we solved the atomic structure of Ad5 E4-ORF3. In Aim 1, we will use this to reveal novel structural motifs that silence p53 target genes. We have discovered that E4-ORF3 proteins from other Adenovirus subgroups do not inactivate p53. This provides a rational basis to identify Ad5 E4-ORF3 specific motifs that silence p53 target genes and engineer viruses with discrete E4-ORF3 mutations that prevent p53 inactivation. In Aim 2, we will combine E4ORF3 and E1B-55K mutations to develop novel Ad5 and Ad34 viruses and test their p53 selectivity in panels of normal and tumor cells. These studies will identify the critical functions of p53 that prevent viral replication and if they are disrupted by p53 and p14ARF mutations in cancer. Finally, in Aim 3, we will test novel p53 selective oncolytic viruses in preclinical mouse models of cancer. We will exploit new technologies to engineer additional genomic modifications that prevent viral uptake in the liver and limiting inflammation, enabling systemic delivery and expanded tumor tropisms. These viral therapies have the potential to be self-perpetuating, kill tumors through regulated cell death, and produce progeny that spread from within the tumor to distant micro-metastases. If results in pre-clinical models are promising, the goal is to test these agents in patients.