Defining Critical p53 Therapeutic Targets and Mechanisms

定义关键的 p53 治疗靶点和机制

• 批准号: 8818821
• 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/8818821
• 关键词: Adenovirus Infections; Adenoviruses; Binding; Binding Sites; CDKN2A gene; Cancer Patient; Cell Death; Cell Nucleus; Cells; Clinical; Clinical Trials; Combined Modality Therapy; DNA Binding; Development; Distant; Distant Metastasis; Dominant-Negative Mutation; Engineered Gene; Engineering; Evolution; Gene Targeting; Genetic Transcription; Genomics; Goals; Grant; Heat-Shock Proteins 90; Heterochromatin; Histone Deacetylase Inhibitor; Human; Immunity; Inflammation; Left; 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; Technology; Testing; Treatment Efficacy; Tropism; Viral; Viral Genome; Viral Physiology; Virus; Virus Inactivation; Xenograft Model; base; cancer therapy; chemotherapy; 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; 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）：本次更新的目标是利用对灭活 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。几乎所有类型的人类癌症中，p53 通路都会因突变而失活。然而，尽管如此，仍然没有经过批准的针对 p53 缺陷肿瘤细胞的合理疗法。腺病毒 E1B-55K 以 p53 为目标进行降解，这被认为对于 p53 失活和病毒复制至关重要。在此前提下，一种名为 ONYX-015 的 B-55K 病毒作为 p53 癌症疗法在临床试验中进行了测试。然而，患者的反应与其肿瘤的 p53 状态无关。虽然 p53 被诱导，但它不活跃。因此，p53 并不决定 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，那么 p53 选择性的巨大潜力仍然可以实现。这是上一个资助期的目标。这导致人们发现Ad5编码另一种蛋白质E4-ORF3，它通过形成核聚合物来使p53失活，该核聚合物指定p53靶启动子的从头异染色质沉默，从而阻止p53-DNA结合。当访问被拒绝时，p53 无法激活下游效应子的转录来限制病毒复制。这些研究改变了 p53 在腺病毒感染中如何失活的长期定义。在这里，我们建议利用这些机制见解来设计 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开。先前的研究揭示了满足这些标准的 E1B-55K H260A 突变。为了识别 E4-ORF3 中的类似突变，我们解析了 Ad5 E4-ORF3 的原子结构。在目标 1 中，我们将利用它来揭示沉默 p53 靶基因的新颖结构基序。我们发现来自其他腺病毒亚群的 E4-ORF3 蛋白不会使 p53 失活。这为鉴定 Ad5 E4-ORF3 特异性基序提供了合理的基础，这些基序可沉默 p53 靶基因，并设计具有离散 E4-ORF3 突变的病毒，以防止 p53 失活。在目标 2 中，我们将结合 E4ORF3 和 E1B-55K 突变来开发新型 Ad5 和 Ad34 病毒，并在正常细胞和肿瘤细胞组中测试它们的 p53 选择性。这些研究将确定 p53 阻止病毒复制的关键功能，以及它们是否会被癌症中的 p53 和 p14ARF 突变所破坏。最后，在目标 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，以防止肝脏中的病毒摄取并限制炎症，从而实现全身递送并扩大肿瘤趋向性。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 产生从肿瘤内部扩散到远处微转移的后代。如果临床前模型的结果很有希望，那么目标就是在患者身上测试这些药物。