Defining Critical p53 Therapeutic Targets and Mechanisms

定义关键的 p53 治疗靶点和机制

• 批准号: 8986160
• 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/8986160
• 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; Health; 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; TP53 gene; Technology; Testing; Treatment Efficacy; Tropism; Viral; Viral Genome; Viral Physiology; Virus; Virus Replication; 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; 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抑制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通路在几乎所有形式的人类癌症中都被突变灭活。然而，尽管如此，仍然没有批准的靶向p53缺陷肿瘤细胞的合理疗法。腺病毒E1 B-55 K靶向p53进行降解，这被认为是p53失活和病毒复制所必需的。在此前提下，一种名为ONYX-015的β B-55 K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，但它是无活性的。因此，p53不决定ONYX-015的肿瘤选择性复制。因此，如果确定腺病毒灭活p53的E1 B-55 K非依赖性机制，则仍然可以实现p53选择性的巨大潜力。这是上一个赠款期的目标。这导致发现Ad 5编码另一种蛋白质E4-ORF 3，其通过形成指定p53靶启动子的从头异染色质沉默的核聚合物使p53失活，从而防止p53-DNA结合。由于通路被拒绝，p53无力激活下游效应子的转录以限制病毒复制。这些研究改变了长期以来对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，工程E1 B-55 K/E4-ORF 3突变腺病毒作为令人兴奋的新的代理p53选择性溶瘤病毒治疗。这需要E1 B-55 K和E4-ORF 3突变，选择性地将p53失活与病毒复制中的其他功能解偶联。先前的研究显示，E1 B-55 K H260 A突变符合这些标准。为了鉴定E4-ORF 3中的类似突变，我们解析了Ad 5 E4-ORF 3的原子结构。在目标1中，我们将利用这一点来揭示沉默p53靶基因的新结构基序。我们已经发现来自其他腺病毒亚组的E4-ORF 3蛋白不与p53结合。这为鉴定使p53靶基因沉默的Ad 5 E4-ORF 3特异性基序和具有防止p53失活的离散E4-ORF 3突变的工程病毒提供了合理的基础。在目标2中，我们将结合联合收割机E4 ORF 3和E1 B-55 K突变来开发新型Ad 5和Ad 34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p14 ARF突变破坏。最后，在目标3中，我们将在癌症的临床前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，以防止肝脏中的病毒摄取并限制炎症，从而实现全身递送和扩大肿瘤向性。这些病毒疗法具有自我延续的潜力，通过调节细胞死亡杀死肿瘤， 产生从肿瘤内扩散到远处微转移的后代。如果临床前模型的结果是有希望的，目标是在患者中测试这些药物。