Defining Critical p53 Therapeutic Targets and Mechanisms

定义关键的 p53 治疗靶点和机制

• 批准号: 9180687
• 负责人: Clodagh O'Shea
• 金额: $ 43.65万
• 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 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9180687
• 关键词: Adenovirus Infections; Adenoviruses; Binding; Binding Sites; CDKN2A gene; Cancer Model; Cancer Patient; Cell Death; Cell Nucleus; Cells; Clinical; Clinical Trials; Combined Modality Therapy; DNA Binding; Development; Distant; Distant Metastasis; Dominant-Negative Mutation; Engineering; Evolution; Gene Targeting; Genetic Transcription; Genomics; Goals; Grant; Heat-Shock Proteins 90; Heterochromatin; Histone Deacetylase Inhibitor; Human; Immunity; Inflammation; Liver; MDM2 gene; Malignant Neoplasms; Mediating; Modification; Mus; Mutagenesis; Mutation; NBS1 gene; Neoplasm Metastasis; New Agents; Normal Cell; Nuclear; ONYX-015; Oncolytic; Oncolytic viruses; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphorylation; Point Mutation; Polymers; Pre-Clinical Model; Protein p53; Proteins; RNA; RNA Interference; Reporter; Specific qualifier value; Structure; Subgroup; TP53 gene; Technology; Testing; Treatment Efficacy; Tropism; Viral; Viral Genome; Viral Physiology; Virus; Virus Replication; Xenograft Model; cancer therapy; chemotherapy; genotoxicity; improved; in vivo; inhibitor/antagonist; insight; killings; mouse model; mutant; neoplastic cell; new technology; new therapeutic target; novel; pre-clinical; prevent; promoter; public health relevance; receptor; response; small hairpin RNA; targeted treatment; therapeutic target; tumor; uptake

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.

描述(由申请人提供)：此次更新的目标是利用对灭活P53的病毒机制的新见解来开发有效的P53肿瘤选择性溶瘤腺病毒疗法。几乎所有形式的人类癌症中的突变都会使P53途径失活。然而，尽管如此，仍然没有公认的针对p53缺陷肿瘤细胞的合理治疗方法。腺病毒E1B-55K以P53为靶点进行降解，这被认为是P53失活和病毒复制的关键。在这一前提下，�B-55K病毒ONYX-015作为一种P53癌症疗法进行了临床试验。然而，患者的反应与他们肿瘤的P53状态无关。虽然P53被诱导，但它是不活跃的。因此，P53不决定Onyx-015的肿瘤选择性复制。因此，如果确定了腺病毒灭活P53的E1B-55K独立机制，仍然可以实现P53选择性的巨大潜力。这是上一次赠款期间的目标。这导致发现Ad5编码另一种蛋白质E4-ORF3，它通过形成一种核聚合物来灭活P53，该核聚合物指定从头开始异染色质沉默P53靶启动子，从而阻止P53与DNA的结合。由于访问被拒绝，p53无法激活下游效应器的转录来限制病毒复制。这些研究改变了长期以来对腺病毒感染中p53如何失活的定义。在这里，我们建议利用这些机制的洞察力来设计E1B-55K/E4-ORF3突变腺病毒，作为P53选择性溶瘤病毒治疗的激动剂。这需要E1B-55K和E4-ORF3突变，选择性地将P53失活与病毒复制中的其他功能分开。先前的研究显示，符合这些标准的E1B-55K H260A突变。为了鉴定E4-ORF3中的类似突变，我们解算了Ad5 E4-ORF3的原子结构。在目标1中，我们将利用这一点来揭示沉默p53靶基因的新的结构基序。我们已经发现，来自其他腺病毒亚群的E4-ORF3蛋白不会使P53失活。这为确定沉默P53靶基因的Ad5E4-ORF3特异基序和利用离散的E4-ORF3突变防止P53失活的工程病毒提供了合理的基础。在目标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组中测试它们的P53选择性。这些研究将确定阻止病毒复制的p53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，在目标3中，我们将在临床前肿瘤小鼠模型中测试新型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摄取，限制炎症，实现全身递送和扩大肿瘤的趋向性。这些病毒疗法有可能自我延续，通过调节细胞死亡来杀死肿瘤，以及 产生从肿瘤内扩散到远处微转移的后代。如果在临床前模型中的结果是有希望的，目标是在患者身上测试这些药物。