Bypassing mutant p53 in radiation therapy

放射治疗中绕过突变型 p53

• 批准号: 9068886
• 负责人: Hongdau Peter Liu
• 金额: $ 4.86万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-15 至 2017-06-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9068886
• 关键词: Aftercare; Apoptosis; Apoptotic; Biological Markers; Blinded; Bypass; CASP2 gene; Cancer Patient; Cancerous; Cell Culture Techniques; Cell Cycle Arrest; Cell Death; Cells; Cleaved cell; Clinical; DNA; DNA Damage; Embryo; FDA approved; Fishes; Genetic; Genetic Determinism; Genetic study; Health; Hela Cells; Human; Ionizing radiation; Libraries; Malignant Neoplasms; Mediating; Methods; Modeling; Mutate; Mutation; PTEN gene; Pathway interactions; Pharmaceutical Preparations; Phenotype; Preclinical Drug Evaluation; Process; Radiation; Radiation Tolerance; Radiation therapy; Radiation-Sensitizing Agents; Radioresistance; Radiosensitization; Regulation; Research; Resistance; Signal Transduction; Specificity; TP53 gene; Testing; Therapeutic; Tumor Suppressor Proteins; Zebrafish; base; cancer cell; cancer therapy; chemical genetics; chemotherapy; clinically relevant; improved; in vivo; inhibitor/antagonist; irradiation; knock-down; mutant; neoplastic cell; novel; novel therapeutic intervention; oncology; patient stratification; protein complex; radiosensitizing; resistance mechanism; response; screening; small molecule; small molecule libraries; targeted treatment; tumor

DESCRIPTION (provided by applicant): Mutation of the tumor suppressor TP53 leads to the evasion of apoptosis after radiation, underlying resistance to conventional cancer treatments, such as radiation and chemotherapy. Our research seeks to bypass these alterations through the discovery of parallel cell-death pathways. We previously discovered a novel apoptotic process, termed "Chk1-suppressed" (CS) pathway, which restores radiosensitivity in TP53 mutant cells after treatment with Chk1 inhibitors (Sidi et al. Cell 2008)1. Using both zebrafish and mammalian TP53-mutant models, our lab has established that the CS pathway requires the assembly of a protein complex called the PIDDosome, composed of PIDD, RAIDD, and caspase-2 (Ando et al. Mol Cell 2012)2. This proposal focuses on (1) identifying further genetic determinants predicting Chk1 inhibitor efficacy in radiotherapy and (2) discovering novel radiosensitizers against tumors with TP53 mutation. Utilizing both human cancer cells and zebrafish, we investigated the impact of cancer alterations other than mutant p53 on cellular sensitivity to CS apoptosis. We found that a mutation in PTEN, the second most commonly mutated tumor suppressor, blocks the execution of apoptosis after ionizing radiation (IR) and Chk1 inhibition (Chk1i). Furthermore, we identified the PTEN-Akt-IAP signaling axis as a candidate pathway to promote resistance to CS apoptosis. The loss of PTEN appears to inhibit the CS pathway downstream of caspase-2 activation, as HeLa cells with impaired PTEN fail to engage in apoptosis, despite an unaffected ability to cleave caspase-2 following IR+Chk1i. Our hypothesis is that one or more IAPs act downstream of Akt to directly inhibit active caspase-2. I propose to test this hypothesis using a candidate knockdown approach to determine which IAP(s), if any, is responsible for caspase-2 inhibition. The findings will be extended in vivo through both chemical and genetic studies in zebrafish. To identify novel targeted strategies for restoring radiosensitivity in TP53 mutant tumors, we exploited the high-throughput drug screening capability of zebrafish to perform a blinded phenotypic radiosensitization screen of 640 FDA-approved compounds. A primary screen of drugs treated in combination with 15 Gy IR yielded 140 compounds capable of producing phenotypes comparable to Chk1i (i.e. >75% sick or dead fish 4 days post irradiation) in p53 mutant fish. Our secondary screen of irradiated vs non-irradiated embryos so far has identified 6 compounds that demonstrate specific radiosensitizing effects. The genetic specificity of these candidate radiosensitizers will be confirmed and prioritized by clinical relevance, proposed mechanism of action, and efficacy. The proposed targets, along with the mechanism(s) of cell death will be confirmed in cell culture and in vivo. The results of these findings may aid in rapidly benefiting cancer patients by proposing a new application for an already approved and available drug.

描述（由申请人提供）：肿瘤抑制因子 TP53 的突变导致放射后细胞凋亡的逃避，从而对传统癌症治疗（例如放射和化学疗法）产生潜在抵抗。我们的研究试图通过发现平行的细胞死亡途径来绕过这些改变。我们之前发现了一种新的凋亡过程，称为“Chk1 抑制”(CS) 途径，该途径在用 Chk1 抑制剂处理后恢复 TP53 突变细胞的放射敏感性 (Sidi et al. Cell 2008)1。使用斑马鱼和哺乳动物 TP53 突变模型，我们的实验室已确定 CS 途径需要组装称为 PIDDosome 的蛋白质复合物，该复合物由 PIDD、RAIDD 和 caspase-2 组成（Ando 等人，Mol Cell 2012）2。该提案的重点是 (1) 进一步确定预测 Chk1 抑制剂在放疗中疗效的遗传决定因素，以及 (2) 发现针对 TP53 突变肿瘤的新型放射增敏剂。 利用人类癌细胞和斑马鱼，我们研究了突变 p53 以外的癌症改变对细胞对 CS 凋亡敏感性的影响。我们发现，第二个最常见突变的肿瘤抑制基因 PTEN 的突变，继电离辐射 (IR) 和 Chk1 抑制 (Chk1i) 之后，阻碍了细胞凋亡的执行。此外，我们确定 PTEN-Akt-IAP 信号轴作为促进 CS 细胞凋亡抵抗的候选途径。 PTEN 的缺失似乎会抑制 caspase-2 激活的 CS 通路下游，因为 PTEN 受损的 HeLa 细胞无法参与细胞凋亡，尽管 IR+Chk1i 后裂解 caspase-2 的能力不受影响。我们的假设是一种或多种 IAP 在 Akt 下游发挥作用，直接抑制活性 caspase-2。我建议使用候选敲低方法来测试这一假设，以确定哪些 IAP（如果有）负责 caspase-2 抑制。这些发现将通过斑马鱼的化学和遗传学研究在体内得到扩展。 为了确定恢复 TP53 突变肿瘤放射敏感性的新靶向策略，我们利用斑马鱼的高通量药物筛选能力对 640 种 FDA 批准的化合物进行盲法表型放射增敏筛选。与 15 Gy IR 联合处理的药物的初步筛选产生了 140 种化合物，这些化合物能够在 p53 突变鱼中产生与 Chk1i 相当的表型（即照射后 4 天，>75% 患病或死亡的鱼）。迄今为止，我们对经过辐射和未经辐射的胚胎进行的二次筛选已经鉴定出 6 种具有特定放射增敏作用的化合物。这些候选放射增敏剂的遗传特异性将根据临床相关性、拟议的作用机制和功效进行确认和优先排序。所提出的目标以及细胞死亡的机制将在细胞培养物和体内得到证实。这些发现的结果可能会通过提出一种方法来帮助癌症患者迅速受益 已批准且可用的药物的新申请。